Polymorphic forms of ST-246 and methods of preparation

ABSTRACT

Polymorph forms of 4-trifluoromethyl-N-(3,3a,4,4a,5,5a,6,6a-octahydro-1,3-dioxo-4,6-ethenocycloprop[f]isoindol-2(1H)-yl)-benzamide are disclosed as well as their methods of synthesis and pharmaceutical compositions.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. patentapplication Ser. No. 16/025,057 filed Jul. 2, 2018, which is adivisional application of U.S. patent application Ser. No. 15/661,194filed Jul. 27, 2017, now U.S. Pat. No. 10,045,964, which is a divisionalapplication of U.S. Patent application Ser. No. 14/959,180, filed onDec. 4, 2015, now U.S. Pat. No. 9,744,154, which is a divisionalapplication of U.S. patent application Ser. No. 13/069,813, filed onMar. 23, 2011, now U.S. Pat. No. 9,339,466 which claims benefit of U.S.Provisional Application No. 61/316,747, filed on Mar. 23, 2010 and U.S.Provisional Application No. 61/373,031, filed on Aug. 12, 2010, thesubject matter of each of which is incorporated by reference in theirentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with U.S. government support under Contract No.HHSN266200600014C awarded by the National Institutes of Health (NIH).The US government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to particular crystalline forms of apharmaceutical compound,4-trifluoromethyl-N-(3,3a,4,4a,5,5a,6,6a-octahydro-1,3-dioxo-4,6-ethenocycloprop[f]isoindol-2(1H)-yl)-benzamide,named ST-246, to processes for their preparation, pharmaceuticalcomposition comprising different crystalline forms and its use intherapy.

BACKGROUND OF THE INVENTION

Throughout this application, various publications are referenced withinthe text. The disclosure of these publications in their entireties arehereby incorporated by reference into this application in order to morefully describe the state of the art as known to those skilled in thereinas of the date of the invention described and claimed herein.

Following the eradication of smallpox (Fenner et al., The epidemiologyof smallpox. In: Smallpox and its eradication. Switzerland: World HealthOrganization; 1988) and the subsequent cessation of routine childhoodvaccinations for smallpox, the number of people susceptible to infectionwith variola virus (VARV), the etiologic agent that causes smallpox, hasdramatically increased worldwide. In addition, encroachment intowildlife habitats, the trade of exotic pets, and the trade of bush meatincrease the risk for zoonotic infection with other orthopoxviruses,such as monkeypox virus (MPXV), for which vaccination against smallpoxprovides some cross protection (Jezek et al., Human monkey pox. In:Melnick J L ed. Monographs in virology. Vol. 17. Basel, Switzerland: SKarger A G. 1988:81-102).

Given that a large proportion of the worldwide population is susceptibleto smallpox, the emergence of MPXV in the United States in 2003, and thecontinued concern over the intentional release of VARV, there is renewedinterest in the development of safer smallpox and other orthopoxvirusvaccines and antiviral therapeutics.

One recently discovered antiviral compound is ST-246, a specific andpotent inhibitor of an orthopoxvirus protein critical for virusmaturation. Several studies evaluating ST-246 for activity againstorthopoxviruses have demonstrated excellent in vitro and in vivoefficacy (Quenelle et al. 2007. Efficacy of delayed treatment withST-246 given orally against systemic orthopoxvirus infections in mice.Antimicrobial Agents and Chemotherapy Feb; 51(2):689-95, Smee et al,2008. Progress in the discovery of compounds inhibiting orthopoxvirusesin animal models. Antiviral Chemistry and Chemotherapy 19(3):115-24).When evaluated in vitro against vaccinia virus (VV), cowpox virus (CV),ectromelia virus (ECTV), monkeypox, camelpox, and variola viruses,ST-246 inhibited virus replication by 50% (50% effective concentration[EC₅₀]) at a concentration of <0.07 μM. With animal models using lethalinfections with ECTV, VV, or CV, ST-246 was reported to be nontoxic andhighly effective in preventing or reducing mortality even whentreatments were delayed up to 72 h post-viral inoculation (Quenelle etal., 2007. Efficacy of delayed treatment with ST-246 given orallyagainst systemic orthopoxvirus infections in mice. Antimicrobial Agentsand Chemotherapy Feb; 51(2):689-95, Smee et al. 2008. Progress in thediscovery of compounds inhibiting orthopoxviruses in animal models.Antiviral Chemistry and Chemotherapy 19(3):115-24). ST-246 was alsoevaluated with the nonlethal mouse tail lesion model using intravenousVV. When ST-246 was administered orally twice a day at 15 or 50 mg/kg ofbody weight for 5 days, the tail lesions were significantly reduced(Smee et al., 2008. Progress in the discovery of compounds inhibitingorthopoxviruses in animal models. Antiviral Chemistry and Chemotherapy19(3):115-24). Most recently, an infant was given ST-246 as anFDA-authorized emergency treatment for eczema vaccinatum which developedafter exposure to the parent's predeployment military smallpoximmunization (Vora et al., 2008, Severe eczema vaccinatum in a householdcontact of a smallpox vaccine. Clinical Infectious Disease 15;46(10):1555-61).

ST-246 was disclosed in WO 2008/130348, WO 2004/112718 and WO2008/079159 as one of the tetracyclic acylhydrazide compounds fortreatment or prophylaxis of viral infections and diseases associatedherewith, particularly those viral infections and associated diseasescaused by the orthopoxvirus. These publications disclose a process forthe preparation of ST-246 but do not disclose what polymorphic form ismade. Nonetheless, the disclosed process yields ST-246 hemihydrate, thepolymorphic Form V as discussed herein below.

The process of making a monohydrate of ST-246 was disclosed in CN101445478A. The data shown in this publication corresponds topolymorphic Form III according to the present classification ofpolymorphs of ST-246.

It has now been unexpectedly discovered that ST-246 can exist in manydifferent polymorphic forms. A particular crystalline form of a compoundmay have physical properties that differ from those of other polymorphicforms and such properties may influence markedly the physico-chemicaland pharmaceutical processing of the compound, particularly when thecompound is prepared or used on a commercial scale. Such differences mayalter the mechanical handling properties of the compound (such as theflow characteristics of the solid material) and the compressioncharacteristics of the compound. Further, the discovery of newpolymorphic forms of such pharmaceutically important compound as ST-246,provides a new opportunity to improve the performance characteristics ofa pharmaceutical end product and enlarges the repertoire of materialsthat a formulation scientist has available for designing, for example, apharmaceutical dosage form of a drug with targeted release profile orother desired physico-chemical properties.

Further, given that new polymorphic forms of a drug substance maydisplay different melting point, hygroscopicity, stability, solubilityand/or dissolution rate, crystallinity, crystal properties,bioavailability, toxicity and formulation handling characteristics,which are among the numerous properties that need to be considered inpreparing medicament that can be effectively administered. Furthermore,regulatory agencies require a definitive knowledge, characterization andcontrol of the polymorphic form of the active component in solidpharmaceutical dosage forms. Thus, there is a need in the art forcrystallization and characterization of new polymorphic forms of ST-246.

SUMMARY OF THE INVENTION

The present invention provides a polymorph Form I of ST-which shows anX-ray powder diffraction pattern having characteristic peaks of about7.63, 10.04, 11.47, 14.73, 15.21, 15.47, 16.06, 16.67, 16.98, 18.93,19.96, 20.52, 20.79, 22.80, 25.16, 26.53, 27.20, 27.60, 29.60, 30.23,30.49, 30.68, 31.14, 33.65, 34.33, 35.29, 35.56, 36.30, 37.36, 38.42,38.66 degrees.

The present invention also provides a polymorph Form II of ST-246 whichshows a X-ray powder diffraction pattern having characteristic accordingto FIG. 2.

The present invention further provides a polymorph Form III of ST-246which shows an X-ray powder diffraction pattern having characteristicpeaks of about 6.71, 9.05, 12.49, 13.03, 13.79, 14.87, 15.72, 16.26,16.74, 18.10, 18.43, 19.94, 21.04, 21.51, 23.15, 23.51, 25.32, 26.24,26.87, 27.32, 27.72, 28.5 5, 29.08, 29.50, 29.84, 31.27, 33.48, 35.36,39.56 degrees.

The present invention also provides a polymorph Form IV of ST-246 whichshows an X-ray powder diffraction pattern having characteristic as shownin FIG. 4.

The present invention further provides a polymorph Form VI ST-246 whichshows an X-ray powder diffraction pattern having characteristic peaks asshown in FIG. 6.

The present invention also provides pharmaceutical compositionscomprising each of the ST-246 polymorphs Forms I-VI and furthercomprising one or more pharmaceutically acceptable carriers, excipients,diluents, additives, fillers, lubricants or binders.

The present invention further provides methods of treating orthopoxvirusinfections or eczema vaccinatum comprising administering to a subjectanimal or human in need thereof a therapeutically effective amount ofeach of the ST-246 polymorphs Forms I-VI.

The present invention also provides methods for the synthesis of each ofthe ST-246 polymorphs Forms I-VI.

The present invention also provides a dosage unit form for oraladministration, wherein ST-246 has a D90% particle size diameter of upto about 300 microns. In some embodiments, ST-246, polymorph I, II, III,IV and VI has a D90% particle size diameter of about 5 microns, in otherembodiments, the D90% particle size diameter is about 16.6 microns, inyet another embodiment, a D90% particle diameter is about 26.6 micronsand in yet another embodiment, the D90% particle diameter is aboutmicrons.

In another aspect of the invention, a unit dosage form for oraladministration comprising 200 mg of ST-246, wherein ST-246 is selectedfrom a group consisting of ST-246 polymorph Form II, ST-246 polymorphForm III, ST-246 polymorph Form IV and ST-246 polymorph Form VI andfurther comprising 33.15 mg of lactose monohydrate; 42.90 mg ofcroscarmellose sodium; 1.95 mg of colloidal silicon dioxide; 13.65 mg ofhypromellose, 7.8 mg of sodium lauryl sulfate; 1.95 mg of magnesiumstearate; and a quantity of microcrystalline cellulose up to 88.60 mgsuch that the total weight of the dosage form, including any impurities,water and residual solvents, is 390 mg.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an X-ray powder diffraction (XRPD) pattern of Form I.

FIG. 2 shows three X-ray powder diffraction (XRPD) patterns of Form II(from three different samples).

FIG. 3 shows an X-ray powder diffraction (XRPD) pattern of Form III.

FIG. 4 shows two X-ray powder diffraction (XRPD) patterns of Form IV(from two different samples).

FIG. 5 shows an X-ray powder diffraction (XRPD) pattern of Form V.

FIG. 6 shows two an X-ray powder diffraction (XRPD) patterns of Form VI(from two different samples).

FIG. 7 depicts Fourier transform infra red (FTIR) spectrum of Form I.

FIG. 8 depicts Fourier transform infra red (FTIR) spectrum of Form III.

FIG. 9 depicts Fourier transform infra red (FTIR) spectrum of Form V.

FIGS. 10, 11, 12 and 13, depict magnified view of FTIR Spectra of Form I(upper panel), Form V (middle panel) and Form III (lower panel).

FIG. 14 depicts XRPD Pattern of Micronized (upper pattern) andunmicronized (lower pattern) Form I.

FIG. 15 depicts XRPD Pattern of micronized (top pattern) andunmicronized (middle and lower patterns from 2 different samples) FormIII.

FIG. 16 depicts effect of particle size on dissolution of 200 mg ST-246Form I capsules with 3% HDTMA wherein the dissolution conditions are 900ml, 0.05M Phosphate buffer, pH 7.5, USP 2 at 75 RPM, 37° C. and thecapsule is made from Form I APIs with particle size D90 of about 5.5microns.

FIG. 17 depicts effect of particle size on dissolution of 200 mg ST-246Form I capsules with 3% HDTMA wherein the dissolution conditions are 900ml, 0.05M Phosphate buffer, pH 7.5, USP 2 at 75 RPM, 37° C. and thecapsule is made from Form I APIs with particle size D90 of about 16.73microns.

FIG. 18 depicts effect of particle size on dissolution of 200 mg ST-246Form I capsules with 3% HDTMA wherein the dissolution conditions are 900ml, 0.05M Phosphate buffer, pH 7.5, USP 2 at 75 RPM, 37° C. and thecapsule is made from Form I APIs with particle size D90 of about 26.55microns.

FIG. 19 depicts effect of particle size on dissolution of 200 mg ST-246Form I capsules with 3% HDTMA wherein the dissolution conditions are 900ml, 0.05M Phosphate buffer, pH 7.5, USP 2 at 75 RPM, 37° C. and thecapsule is made from Form I APIs with particle size D90 is about 75microns.

FIG. 20 depicts effect of particle size on dissolution of 200 mg ST-246Form I capsules with 3% HDTMA wherein the dissolution conditions are 900ml, 0.05M Phosphate buffer, pH 7.5, USP 2 at 75 RPM, 37° C. and thecapsule is made from Form I APIs with particle size D90 of about 254microns.

FIG. 21 depicts dissolution profile of Form I.

FIG. 22 depicts dissolution profile of Form III.

FIG. 23 depicts dissolution profile of Form V.

FIG. 24 depicts the mean (SD) ST-246 plasma concentrations over time (PKpopulation) after a single oral administration.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In accordance with this detailed description, the followingabbreviations and definitions apply. It must be noted that as usedherein, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise.

The term “polymorphic form, polymorph, polymorph form, crystalline form,physical form or crystalline polymorph” of ST-246 in the presentinvention refers to a crystal modification of ST-246, which can becharacterized by analytical methods such as X-ray powder diffractionpattern, (XRPD), differential scanning calorimetry (DSC), by its meltingpoint analysis or Infrared Spectroscopy (FTIR).

The term “hydrate” as used herein means a compound of the invention or asalt thereof that further includes a stoichiometric ornon-stoichiometric amount of water bound by non-covalent intermolecularforces. Hydrates are formed by the combination of one or more moleculesof water with one molecule of the substances in which the water retainsits molecular state as H₂O, such combination being able to form one ormore hydrate. The term “hemihydrate” as used herein refers to a solidwith 0.5 molecule of H₂O per molecule of the substance.

The term “pharmaceutical composition” or “pharmaceutical formulation” isintended to encompass a drug product including the active ingredient(s),pharmaceutically acceptable excipients that make up the carrier, as wellas any product which results, directly or indirectly, from combination,complexation or aggregation of any two or more of the ingredients.Accordingly, the pharmaceutical compositions of the present inventionencompass any composition made by admixing the active ingredient, activeingredient dispersion or composite, additional active ingredient(s), andpharmaceutically acceptable excipients.

The particle size distribution (PSD) of a powder, or granular material,or particles dispersed in fluid, is a list of values or a mathematicalfunction that defines the relative amounts of particles present, sortedaccording to size. PSD is also known as grain size distribution. Sinceparticle size for a complex media is a distribution of diameters,statistics can be used to convey the results. A common method is to used10, d50 and d90 values based on volume distribution. That is to saythat 10%, 50% and 90%, respectively, of the particle size distributionis smaller than the stated diameter.

The term “dosage unit” refers to a single unit of the dosage form thatis to be administered to the patient. The dosage unit will be typicallyformulated to include an amount of drug sufficient to achieve atherapeutic effect with a single administration of the dosage unitalthough where the size of the dosage form is at issue, more than onedosage unit may be necessary to achieve the desired therapeutic effect.For example, a single dosage unit of a drug is typically, one tablet,one capsule, or one tablespoon of liquid. More than one dosage unit maybe necessary to administer sufficient drug to achieve a therapeuticeffect where the amount of drug causes physical constraints on the sizeof the dosage form.

The term “half-life” is a pharmacokinetic term used to indicate thelength of time necessary to eliminate 50% of the remaining amount ofdrug present in the body.

The term “AUC” (i.e., “area under the curve,” “area under theconcentration curve,” or “area under the concentration-time curve”) is apharmacokinetic term used to refer a method of measurement ofbioavailability or extent of absorption of a drug based on a plot of anindividual or pool of individual's blood plasma concentrations sampledat frequent intervals; the AUC is directly proportional to the totalamount of unaltered drug in the patient's blood plasma. For example, alinear curve for a plot of the AUC versus dose (i.e., straight ascendingline) indicates that the drug is being released slowly into the bloodstream and is providing a steady amount of drug to the patient; if theAUC versus dose is a linear relationship this generally representsoptimal delivery of the drug into the patient's blood stream. Bycontrast, a non-linear AUC versus dose curve indicates rapid release ofdrug such that some of the drug is not absorbed, or the drug ismetabolized before entering the blood stream.

The term “C_(max)” (i.e., “maximum concentration”) is a pharmacokineticterm used to indicate the peak concentration of a particular drug in theblood plasma of a patient.

The term “T_(max)” (i.e., “time of maximum concentration” or “time ofC_(max)”) is a pharmacokinetic term used to indicate the time at whichthe C_(max) is observed during the time course of a drug administration.As would be expected, a dosage form that would include an immediaterelease as well as a gastric retentive component would have a T_(max)that is higher than the C_(max) for an immediate release dosage form,but lower than the T_(max) for a purely gastric retentive dosage form.

It has now been surprisingly discovered that ST-246 exists in differentcrystalline forms denominated Form I, Form II, Form III, Form IV, FormV, and Form VI.

All forms have been fully characterized and comparability data have beengenerated. All the forms are as characterized hereinafter inter alia bythe following methodology:

Physical Experimental Methodology

X-Ray Powder Diffraction (XRPD)

Diffraction patterns were collected using a Bruker D8 Discoverydiffractometer configured with an XYZ stage, laser video microscope forpositioning, and HiStar area detector. Collection times were nominally60 seconds. A Cu Ka radiation 1.5406 tube was operated at 40 kV and 40mA was used to irradiate samples. The X-ray optics consists of a Gobelmirror coupled with a pinhole collimator of 0.5 mm. Theta-thetacontinuous scans were employed with a sample-detector distance of 15 cm,which gives an effective 20 range of 4-40°. Samples were mounted in lowbackground quartz plates. A variable temperature hot stage was used tomanipulate sample temperature for some experiments.

The polymorphs of ST-246 are characterized by their X-ray powderdiffraction patterns (XRPD) and/or their Raman spectroscopy peaks. Withrespect to X-ray powder diffraction, the relative intensities of theX-ray powder diffraction peaks of a given polymorph may vary dependingupon the crystal size of the polymorph used to determine the pattern.This is a phenomenon of preferred orientation. Preferred orientation iscaused by the morphology of crystals. In this case, the XRPD analysismay be carried out with the sample spinning in the sample holder duringXRPD analysis to reduce the preferred orientation effects. For the XRPDanalysis, the pattern is given in terms of the “degree of 20 (twotheta)” angles of the peaks.

With respect to the percent value of relative intensity (I/lo), Iorepresents the value of the maximum peak determined by XRPD for thesample for all “degree 26.” angles and I represents the value for theintensity of a peak measured at a given “degree 2θ” angle”.

The angle “26 degree” is a diffraction angle which is the angle betweenthe incident X-rays and the diffracted X-rays. The values for therelative intensities for a given peak set forth in percent and the“degree.2θ” angles are calculated. However, there are key major peaks atgiven angles in these X-ray powder diffraction patterns which are uniqueto each given polymorph form. These peaks are present in the XRPDpatterns of each of the polymorph forms having a crystal size of about10 to 40 microns. Any of these major peaks, either alone or in anydistinguishing combination, are sufficient to distinguish one of thepolymorph forms from the other present polymorphic forms.

Infrared Spectroscopy (Ftir)

Infrared spectra were obtained with a Nicolet 510 M-O Fourier transforminfrared spectrometer, equipped with a Harrick Splitpea™ attenuatedtotal reflectance device. Spectra were acquired from 4000-400 cm⁻¹ witha resolution of 4 cm⁻¹ and 128 scans were collected for each analysis.

Preparation of the Crystalline Forms

The present invention provides a method of producing polymorphic Form Iof ST-246, comprising the steps of:

a) dissolving ST-246 in at least one organic solvent and an amount ofwater to make a solution;

b) cooling said solution to a temperature that causes the preferentialcrystallization of said ST-246 polymorphic Form I; and

c) optionally drying the formed crystals of ST-246,

wherein said organic solvent is selected from a group consisting ofisopropyl alcohol (IPA), ethyl acetate, ethanol, methanol, acetone,isopropyl acetate and tetrahydrofuran (THF).

Preferably, the method further comprises adding seed crystals ofpolymorphic Form I ST-246 during step (b). Also preferably, the coolingstep takes place over at least 15 minutes, more preferably over at least2 hours and most preferably over at least 5 hours.

Also preferably, the organic solvent is ethyl acetate and the watercontent is about 40% by volume of total solvent volume, more preferablyabout 5% by volume of total solvent volume, more preferably about 3% byvolume of total solvent volume and most preferably about 2% by volume oftotal solvent volume. Also preferably, the organic solvent is isopropylalcohol and the water content is about 5% by volume of total solventvolume.

The present invention also provides a method of producing crystalpolymorphic Form II of ST-246, comprising the steps of:

a) dissolving ST-246 in at least one solvent to make a solution;

b) cooling said solution to a temperature that causes the preferentialcrystallization of said ST-246 polymorphic Form II; and

c) optionally drying the formed crystals of ST-246,

wherein said solvent is selected from the group consisting of ethylacetate, chloroform, 1-propanol, isopropyl alcohol (IPA) ethanol,acetone, acetonitrile, toluene, isopropyl acetate and dimethylformamide(DMF).

Preferably, the method further comprises adding seed crystals ofpolymorphic Form II ST-246 during step (b). Also preferably, the solventdoes not contain water and is selected from the group consisting ofethyl acetate and chloroform.

The present invention further provides a method of producing crystalpolymorphic Form II of ST-246, comprising the steps of:

a) dissolving ST-246 in ethanol and water to make a solution;

b) cooling said solution to a temperature that causes the preferentialcrystallization of said ST-246 polymorphic Form II; and

c) optionally drying the formed crystals of ST-246.

Preferably, the method further comprises adding seed crystals ofpolymorphic Form II ST-246 during step (b). Also preferably, the volumeratio of ethanol:water is about 1:1.

The present invention also provides a method of producing crystalpolymorphic Form III of ST-246, comprising the steps of:

a) dissolving ST-246 in at least one organic solvent and water to make asolution;

b) cooling said solution to a temperature that causes the preferentialcrystallization of said ST-246 polymorphic Form IIII: and

c) optionally drying the formed crystals of ST-246,

wherein the organic solvent is selected from the group consisting ofisopropyl alcohol (IPA), ethyl acetate and ethanol.

Preferably, the method further comprises adding seed crystals ofpolymorphic Form III ST-246 during step (b). Also preferably, thecooling step takes place over less than 15 minutes.

The present invention further provides a method of producing crystalpolymorphic Form III of ST-246, comprising the steps of:

a) dissolving ST-246 in at least one organic solvent to make a solution;

b) cooling said solution to a temperature that causes the preferentialcrystallization of said ST-246 polymorphic Form III; and

c) optionally drying the formed crystals of ST-246, wherein the organicsolvent is selected from the group consisting of acetone, isopropylalcohol (IPA), dimethylamine (DMA), pyridine, trifluoroethanol (TFE),methanol, ethanol, chloroform, acetonitrile (ACN), and tetrahydrofuran(THF).

Preferably, the method further comprises adding seed crystals ofpolymorphic Form III ST-246 during step (b).

The present invention also provides a method of producing crystalpolymorphic Form IV of ST-246, comprising the steps of:

a) dissolving ST-246 in at least one organic solvent optionallycontaining water to make a solution;

b) cooling said solution to a temperature that causes the preferentialcrystallization of said ST-246 polymorphic Form IV precipitation incrystal form of ST-246; and

c) optionally drying the formed crystals of ST-246,

wherein said solvent is selected from the group consisting of: a mixtureof acetonitrile and ethyl acetate, a mixture of ethanol and toluene, amixture of water and ethyl acetate, and a mixture of trifluoroethanoland THF.

Preferably, the method further comprises adding seed crystals ofpolymorphic Form IV ST-246 during step (b). Also preferably, the solventis a mixture of ACN and ethyl acetate at a volume ratio of about 1:4, amixture of ethanol and toluene at a volume ratio of about 1:4, a mixtureof water and ethyl acetate at a volume ratio of about 1:4, and a mixtureof TFE and THF at a volume ratio of about 1:1.

The present invention further provides a method of producing crystalpolymorphic Form IV of ST-246, comprising the steps of:

a) dissolving ST-246 in at least one solvent to make a solution;

b) cooling said solution to a temperature that causes the preferentialcrystallization of said ST-246 polymorphic Form IV; and

c) optionally drying the formed crystals of ST-246,

wherein said solvent is selected from the group consisting of 1-butanol,trifluoroethanol (TFE), chloroform, dichloromethane and toluene.

Preferably, the method further comprises adding seed crystals ofpolymorphic Form IV ST-246 during step (b). Also preferably, the solventdoes not contain water. Also preferably the solvent is 1-butanol.

The present invention also provides a method of producing crystalpolymorphic Form VI of ST-246, comprising the steps of:

a) dissolving ST-246 in at least one solvent to make a solution;

b) cooling said solution to a temperature that causes the preferentialcrystallization of said ST-246 polymorphic Form VI; and

c) optionally drying the formed crystals of ST-246,

wherein said solvent is selected from the group consisting ofnitromethane, methanol and chloroform.

Preferably, the method further comprises adding seed crystals ofpolymorphic Form VI ST-246 during step (b). Also preferably, the solventdoes not contain water and is nitromethane.

The ST-246 is prepared as outlined in the Examples below. Processes forcrystallization of polymorphs of the ST-246 may embrace multiplecombinations of techniques and variations thereof. Crystallization ofpolymorphs of the ST-246 may be executed by dissolving, dispersing, orslurrying ST-246 at a suitable temperature in the solvent wherebyportion of the said solvent evaporates increasing the concentration ofthe ST-246 in the said solution, dispersion, or slurry, cooling the saidmixture, and optionally washing and/or filtering and drying theresulting crystals of the ST-246.

Crystal formation may as well involve more than one crystallizationprocess. In certain cases, one, two or more extra crystallization stepsmay be advantageously performed for different reasons, such as, toincrease the quality of the resulting crystal form. For instance, thepolymorphs of the present invention could also be prepared by adding asolvent to an initial starting base material of the ST-246, stirring thesolution at a fixed temperature until the substances would be fullydissolved, concentrating the solution by vacuum distillation, andcooling. A first crystallization would take place and the formedcrystals would be washed with a solvent, and followed by solubilizationof the ST-246 with the same or different solvent to form the desiredpolymorph. The reaction mixture may be heated to reflux andrecrystallization of the reaction mixture would occur, followed by acooling step from reflux. The formed polymorph would optionally befiltered and allowed to dry.

By dissolving, dispersing, or slurrying the ST-246 in the solvent, onemay obtain different degrees of dispersion, such as suspensions,slurries or mixtures; or preferably obtain homogeneous one-phasesolutions. The term “suspension” refers to a two-phase system consistingof a finely divided solid, i.e. ST-246 in amorphous, crystalline form,or mixtures thereof, dispersed (suspended) in a liquid or dispersingmedium, usually the solvent. The term “slurry” refers to a suspensionformed when a quantity of powder is mixed into a liquid in which thesolid is only slightly soluble (or not soluble). “Slurrying” refers tothe making of a slurry.

Optionally, the solvent medium may contain additives, for exampledispersing agents, surfactants or other additives, or mixtures thereofof the type normally used in the preparation of crystalline suspensions.The additives may be advantageously used in modifying the shape ofcrystal by increasing the leniency and decreasing the surface area.

The solvent medium containing the solid may optionally be stirred for acertain period of time, or vigorously agitated using, for example, ahigh shear mixer or homogenizer or a combination of these, to generatethe desired particle size for the organic compound. Control ofprecipitation temperature and seeding may be additionally used toimprove the reproducibility of the crystallization process, the particlesize distribution and the dosage form of the product. As such, thecrystallization can be effected without seeding with crystals of theST-246 or preferably in the presence of crystals of the ST-246, whichare introduced into the solution by seeding. Seeding can also beeffected several times at various temperatures. The amount of the seedmaterial depends on the scale of the experiment and can readily bedetermined by a person skilled in the art. Typically, the amount ofseeding material is about 0.1 to 1 weight % of the amount of crystallinematerial expected from the reaction.

The time for crystallization in each crystallization step will depend onthe conditions applied, the techniques employed and/or solvents used.Breaking up the large particles or aggregates of particles after crystalconversion may additionally be performed in order to obtain a desiredand homogeneous particle size. Accordingly, the crystals, powderaggregates and coarse powder of the polymorphic forms of the ST-246 maybe optionally milled and sorted by size after undergoing conversion.Milling or grinding refers to physically breaking up the large particlesor aggregates of particles using methods and apparatus well known in theart for particle size reduction of powders. Resulting particle sizes mayrange from millimeters to nanometers, yielding i.e. nanocrystals,microcrystals. A preferred apparatus for milling or grinding is a fluidenergy mill, or micronizer, because of its ability to produce particlesof small size within a narrow range of particle size distribution.

The invention provides as well a process wherein the obtainedcrystalline form is isolated by filtration or centrifugation, optionallycombined with washing and drying. The starting material used for theprocesses of the present invention may be any crystalline or amorphousform of the ST-246, including a hydrate thereof.

In one aspect of the invention, the solvents employed in the preparationof the crystalline forms of the present invention are pharmaceuticallyacceptable or pharmaceutically non-acceptable solvents. Pharmaceuticallynon-acceptable solvents will have to be removed prior to using thepolymorph into a pharmaceutical formulation.

The processes for the production of the polymorphic forms of the presentinvention typically include obtaining a crystalline solid material froma solution or dispersion of the ST-246 in a solvent medium, or fromslurrying the ST-246, which can be initially in amorphous or crystallineform. The conditions concerning crystallization may be modified in orderto improve the crystallization process or to induce precipitation, andwithout affecting the form of the polymorph obtained. These conditionsinclude bringing the solution, dispersion, or slurry of the ST-246 andthe solvent(s) to a desired concentration, cooling it following adefined cooling/temperature curve, adding crystal seeds, bringing thesaid solution, dispersion, or slurry to a desired temperature, effectingany suitable pressure, removing and/or separating any undesired materialor impurities, drying the formed crystals to obtain the polymorphs in asolid state, if such state is desired.

One way of inducing precipitation is to reduce the solubility of theST-246. The solubility of the compound may be reduced, for example, bycooling the solution. The solubility of the ST-246 may be reduced byadding an anti-solvent. Bringing the solution, dispersion, or slurry ofthe ST-246 and solvents to a desired concentration does not necessarilyimply an increase in the concentration of the ST-246. In certain cases,a decrease or no change in concentration of the ST-246 could bepreferable. The techniques used for obtaining a desired concentrationinclude, for instance, evaporation by atmospheric distillation, vacuumdistillation, fractioned distillation, azeotropic distillation, filmevaporation, heating, cooling, other techniques well known in the artand combinations thereof. An optional process for obtaining a desiredconcentration could as well involve the saturation of the solution ofthe ST-246 and solvent, for example, by adding a sufficient volume of anon-solvent to the solution to reach the saturation point.

Other suitable techniques for saturating the solution include, by way ofexample, the introduction of additional ST-246 to the solution and/orevaporation of a portion of the solvent from the solution. As referredto herein, a saturated solution encompasses solutions at theirsaturation points or exceeding their saturation points, i.e.supersaturated. A nearly saturated solution refers to solutions that arenear saturation but have not reached their saturation solubility limits.

In the preferred aspect of the invention, crystallization solvent is animportant factor in determining which ST-246 polymorph is formed. Watercontent is also important, because the different polymorphic forms havevarying levels of hydration. In the mixtures of water and water misciblesolvents, the amount of water can vary from about 0.1% by volume toabout 95% by volume, preferably from about 10% to about 20% by volume,more preferably from about 5% to about 10% by volume and most preferablyfrom about 5% to about 1% of water.

ST-246 Polymorphic Forms I and III are monohydrates, and thus there is aminimum threshold of water that must be present in order for ST-246 tocrystallize as a monohydrate. In addition, the cooling rate andisolation temperature and amount of water may play a role in determiningwhich ST-246 polymorphic form and/or hydrate is formed. As summarized inTable 1 below, there is a correlation between cooling rate isolationtemperature, water content and generation of ST-246 Form I or ST-246Form III. Further, the data summarized in Table 1 suggests that thesolvent composition, crystallization temperature, or cooling rate, mayhave an impact on ST-246 polymorph Form formation. For example, as shownin Table 1, when both isopropyl alcohol (IPA) and ethyl acetate are usedas the major solvent, ST-246 Form III is generated when a warm, about35° C. to about 40° C., solution containing higher water content iscooled directly into an ice bath. In contrast, in the presence of thelower water content or when solutions were cooled to room temperaturefor isolation, ST-246 Form I is obtained. Polymorphic form of thematerial prior to final crystallization does not impact final polymorphform, as long as the material completely dissolves in thecrystallization solvent.

TABLE 1 Correlation between ST-246 Form formation, solvent system andisolation temperature. Solvent System Isolation Temperature Form IPA, 5%Water Room Temperature I IPA, 5% Water Ice Bath (2-5° C.) III IPA, 2%Water Ice Bath I Ethyl acetate, 5% Water Room Temperature I Ethylacetate, 5% Water Ice Bath (2-5° C.) III Ethyl acetate, 2% Water IceBath (2-5° C.) I

Removing and/or separating any undesired material or impurities may beperformed by purification, filtering, washing, precipitation or similartechniques. Separation, for example, can be conducted by knownsolid-liquid separation techniques. The filtrations can be performed,amongst other methods, by passing the solution, dispersion, or slurrythrough a filter paper, sintered glass filter or other membranematerial, by centrifugation, or using Buchner style filter, Rosenmundfilter or plates, or frame press. Preferably, in-line filtration orsafety filtration may be advantageously intercalated in the processesdisclosed above, in order to increase the polymorphic purity of theresulting crystalline form.

Crystals obtained may be also dried, and such drying process mayoptionally be used in the different crystallization passages, if morethan one crystallization passage is applied. Drying procedures includeall techniques known to those skilled in the art, such as heating,applying vacuum, circulating air or gas, adding a desiccant,freeze-drying, spray-drying, evaporating, or the like, or anycombination thereof.

Form I.

In one aspect of the invention, the crystalline form of ST-246 isdisclosed and is denominated as Form I of the ST-246, or in short “FormI”.

One preferred parameter to reliably crystallize Form I is the use ofethyl acetate/water mixtures. Several parameters have been varied duringcrystallization studies with ethyl acetate/water (amount of water added,dissolving temperature, isolation temperature, cooling rate) and aresummarized in Tables 1 and 2. Form I can be generated with the use ofethyl acetate/water mixture provided enough water is present to allowformation of the monohydrate. Further, Form I has been shown to beformed using THF/water mixtures, IPA/water mixtures, and both acetoneand methanol have shown the ability to crystallize Form I with higherlevels of water. In addition, Form I can also be generated by holding awater slurry of Forms III and V for several days or longer.

TABLE 2 Crystallization Parameters Dissolving Treatment forprecipitation Drying No. Solvent Water % Temp (° C.) Temp (° C.) Time(hours) Temp (° C.) 4 IPA/Water 5 55-60 RT 14 50 5 IPA/Water 5 55-60 RT:Ice bath  14: 2-4 50 8 IPA/Water 2 35-40 Ice bath 22 50 13 IPA/Water 550-55 14 IPA/Water 2.85 55-60 15 IPA/Water 2.85 55-60 16 IPA/Water 2.8555-60 Ethyl Acetate/Water# 3 70-80 RT 20 75 C. 27 Ethyl Acetate/Water 570-80 RT   5.5 50 29 Ethyl Acetate/Water 5 55-60 RT: Ice bath  14: 2-450 30 Ethyl Acetate/Water 2 55-60 RT 14 50 32 Ethyl Acetate/Water 235-40 Ice bath 22 50 36 Ethyl Acetate → Water 5 50-60 RT O/N 50-60 37Ethyl Acetate → Water 10 50-60 RT O/N 50-60 38 Ethyl Acetate → Water 2050-60 RT O/N 50-60 39 Ethyl Acetate → Water 40 50-60 RT O/N 50-60 40Ethyl Acetate — 55-60 41 Ethyl Acetate/Water 2 55-60 42 EthylAcetate/Water 3 55-60 43 Ethyl Acetate/Water 2 55-60 44 EthylAcetate/Water 3 55-60 45 Ethyl Acetate/Water 2 40-47 46 EthylAcetate/Water 5 40-45 47 Ethyl Acetate/Water 5 40-45 48 EthylAcetate/Water 2 40-45 49 Ethyl Acetate/Water 2 40-45 50 EthylAcetate/Water 2 40-45 62 Ethanol/Water (60° C.) 10 60 RT: 2-8 3: 4 50-6063 Ethanol/Water 3 55-60 64 Ethanol/Water 3 55-60 66 Methanol/Water 555-60 RT: Ice bath  14: 2-4 50 68 Methanol/Water 5 35-40 Ice bath 22 5071 Acetone/Water 7.5 55-60 RT: Ice bath  14: 2-4 50 77 THF/Water 7.555-60 RT: Ice bath  14: 2-4 50 78 THF/Water 2 55-60 RT 14 50 79THF/Water 5 35-40 Ice bath 22 50 80 THF/Water 2 35-40 Ice bath 22 50 83Isopropyl Acetate/Water 5 55-60 RT: Ice bath  14: 2-4 50 84 IsopropylAcetate/Water 2 55-60 RT 14 50 96 Water Slurry 100 45: RT NA 61Ethanol/Water (60° C.) 10 60 RT: 2-8 3: 4 50-60 1 IPA — 70-80 RT 72 75 7IPA/Water 5 35-40 Ice bath 22 50 17 IPA/Water 2.85 55-60 18 IPA/Water2.85 55-60 19 IPA/Water 5 55-60 20 IPA/Water 5 55-60 21 IPA/Water 2.8555-60 22 IPA/Water 5 55-60 23 IPA/Water 5 55-60 24 IPA/Water 1.25 40-4531 Ethyl Acetate/Water 5 35-40 Ice bath 22 50 56 Ethyl Acetate:Hexane(7:4) — 70 RT: 2-8 3: 4 50-60 72 Acetone/Water 2 55-60 RT 15 50 88 WaterSlurry 100 RT: 50 NA 90 Water Slurry 100 NA 91 Water Slurry 100 RT NA RT92 Water Slurry 100 45 NA RT 93 Water Slurry 100 RT NA 37 94 WaterSlurry 100 RT NA 37 95 Water Slurry (14KM71A) 100 60 NA 6 IPA/Water 255-60 RT 14 50 25 Ethyl Acetate — 70-80 RT 72 75 55 Ethyl Acetate:Hexane(7:4) — 70 RT: 2-8 3: 4 50-60 58 Ethanol — 70-80 RT 72 75 67Methanol/Water 2 55-60 RT 15 50 69 Methanol/Water 2 35-40 Ice bath 22 5061 Ethanol/Water (60° C.) 10 60 RT: 2-8 3: 4 50-60 1 IPA — 70-80 RT 7275 7 IPA/Water 5 35-40 Ice bath 22 50 17 IPA/Water 2.85 55-60 18IPA/Water 2.85 55-60 19 IPA/Water 5 55-60 20 IPA/Water 5 55-60 21IPA/Water 2.85 55-60 22 IPA/Water 5 55-60 23 IPA/Water 5 55-60 24IPA/Water 1.25 40-45 31 Ethyl Acetate/Water 5 35-40 Ice bath 22 50 56Ethyl Acetate:Hexane (7:4) — 70 RT: 2-8 3: 4 50-60 72 Acetone/Water 255-60 RT 15 50 88 Water Slurry 100 RT: 50 NA 90 Water Slurry 100 NA 91Water Slurry 100 RT NA RT 92 Water Slurry 100 45 NA RT 93 Water Slurry100 RT NA 37 94 Water Slurry 100 RT NA 37 95 Water Slurry (14KM71A) 10060 NA 6 IPA/Water 2 55-60 RT 14 50 25 Ethyl Acetate — 70-80 RT 72 75 55Ethyl Acetate:Hexane (7:4) — 70 RT: 2-8 3: 4 50-60 58 Ethanol — 70-80 RT72 75 67 Methanol/Water 2 55-60 RT 15 50 69 Methanol/Water 2 35-40 Icebath 22 50 Polymorph Melting Recrystallization/ No. Solvent Form Point(° C.) NB ref. Batch # 4 IPA/Water I 197.9 DN-383-15 383-18-A-1 5IPA/Water 197.8 DN-383-15 383-18-A-1-2 8 IPA/Water 197.7 DN-383-19 13IPA/Water 14KM24C 14 IPA/Water 14KM53D 15 IPA/Water 14KM54B 16 IPA/Water14KM54D Ethyl Acetate/Water# DN-383-1 27 Ethyl Acetate/Water 197.3DN-383-9 29 Ethyl Acetate/Water 198 DN-383-15 383-18-B-1-2 30 EthylAcetate/Water 197.7 DN-383-15 383-18-B-2 32 Ethyl Acetate/Water 197.9DN-383-19 36 Ethyl Acetate → Water MAS-518-88 MAS-518-88-1 37 EthylAcetate → Water MAS-518-88 MAS-518-88-2 38 Ethyl Acetate → WaterMAS-518-88 MAS-518-88-3 39 Ethyl Acetate → Water MAS-518-88 MAS-518-88-440 Ethyl Acetate 14KM40C 41 Ethyl Acetate/Water 14KM40D 42 EthylAcetate/Water 14KM46B 43 Ethyl Acetate/Water 14KM48B 44 EthylAcetate/Water 14KM57E 45 Ethyl Acetate/Water 14KM75B (400 g) 46 EthylAcetate/Water 14KM77A (200 g) 47 Ethyl Acetate/Water 14KM79B (200 g) 48Ethyl Acetate/Water 14KM98B (200 g) 49 Ethyl Acetate/Water 15KM16A (400g) 50 Ethyl Acetate/Water 15KM18B (400 g) 62 Ethanol/Water (60° C.)MAS-518-88 MAS-518-88-6 63 Ethanol/Water 14KM36C 64 Ethanol/Water14KM36D 66 Methanol/Water 197.6 DN-383-15 383-18-C-1-2 68 Methanol/Water197.8 DN-383-19 71 Acetone/Water 199.1 DN-383-15 383-18-D-1-2 77THF/Water DN-383-15 383-18-E-1-2 78 THF/Water DN-383-15 383-18-E-2 79THF/Water DN-383-19 80 THF/Water DN-383-19 83 Isopropyl Acetate/WaterDN-383-15 383-18-F-1-2 84 Isopropyl Acetate/Water DN-383-15 383-18-F-296 Water Slurry 61 Ethanol/Water (60° C.) I and III MAS-518-88MAS-518-88-5 1 IPA III 198.3 DN-383-1 0374-24 7 IPA/Water 197.7DN-383-19 17 IPA/Water 14KM23D 18 IPA/Water 14KM38A 19 IPA/Water 14KM41A20 IPA/Water 14KM49B 21 IPA/Water 14KM60E 22 IPA/Water 14KM64C 23IPA/Water 14KM64D 24 IPA/Water 14KM73B 31 Ethyl Acetate/Water 197.9DN-383-19 56 Ethyl Acetate:Hexane (7:4) MAS-518-88 MAS-518-88-8 72Acetone/Water 197.5 DN-383-15 383-18-D-2 88 Water Slurry 197.5 DN-383-23ST-246W 90 Water Slurry 197.5 91 Water Slurry 197.9 DN-383-34 0383-34 92Water Slurry 197.8 DN-383-34 0383-34 93 Water Slurry WW-386-20 #51 94Water Slurry WW-386-22 #54 95 Water Slurry (14KM71A) 6 IPA/Water V 198.1DN-383-15 383-18-A-2 25 Ethyl Acetate 197.9 DN-383-1 0374-26 55 EthylAcetate:Hexane (7:4) MAS-518-88 MAS-518-88-7 58 Ethanol 197.7 DN-383-10374-28 67 Methanol/Water 197.6 DN-383-15 383-18-C-2 69 Methanol/Water197.9 DN-383-19 61 Ethanol/Water (60° C.) I and III MAS-518-88MAS-518-88-5 1 IPA III 198.3 DN-383-1 0374-24 7 IPA/Water 197.7DN-383-19 17 IPA/Water 14KM23D 18 IPA/Water 14KM38A 19 IPA/Water 14KM41A20 IPA/Water 14KM49B 21 IPA/Water 14KM60E 22 IPA/Water 14KM64C 23IPA/Water 14KM64D 24 IPA/Water 14KM73B 31 Ethyl Acetate/Water 197.9DN-383-19 56 Ethyl Acetate:Hexane (7:4) MAS-518-88 MAS-518-88-8 72Acetone/Water 197.5 DN-383-15 383-18-D-2 88 Water Slurry 197.5 DN-383-23ST-246W 90 Water Slurry 197.5 91 Water Slurry 197.9 DN-383-34 0383-34 92Water Slurry 197.8 DN-383-34 0383-34 93 Water Slurry WW-386-20 #51 94Water Slurry WW-386-22 #54 95 Water Slurry (14KM71A) 6 IPA/Water V 198.1DN-383-15 383-18-A-2 25 Ethyl Acetate 197.9 DN-383-1 0374-26 55 EthylAcetate:Hexane (7:4) MAS-518-88 MAS-518-88-7 58 Ethanol 197.7 DN-383-10374-28 67 Methanol/Water 197.6 DN-383-15 383-18-C-2 69 Methanol/Water197.9 DN-383-19

Form I is a monohydrate crystalline form of ST-246. Examples of X-RayDiffraction (XRPD), data for Form I are summarized in FIG. 1 and areshown below:

Angle d value Intensity 2-Theta Angstrom Cps Intensity % 7.63 11.58 5.925.5 10.04 8.80 35.5 33.3 11.47 7.71 26.8 25.1 14.73 6.01 13.8 12.9 15.215.82 7.67 7.2 15.47 5.72 14.0 13.1 16.06 5.51 20.4 19.1 16.67 5.31 21.520.1 16.98 5.22 9.21 8.6 18.93 4.68 107 100.0 19.96 4.45 29.4 27.5 20.524.32 12.5 11.7 20.79 4.27 48.2 45.2 22.80 3.90 79.6 74.5 25.16 3.54 4.173.9 26.53 3.36 14.0 13.1 27.20 3.28 8.55 8.0 27.60 3.23 9.21 8.6 29.603.02 10.7 10.1 30.23 2.95 48.5 45.4 30.49 2.93 69.5 65.1 30.68 2.91 25.023.4 31.14 2.87 7.67 7.2 33.65 2.66 104 97.3 34.33 2.61 16.9 15.8 35.292.54 10.1 9.4 35.56 2.52 19.5 18.3 36.30 2.47 11.8 11.1 37.36 2.41 32.930.8 38.42 2.34 3.51 3.3 38.66 2.33 28.7 26.9

The characteristic infrared spectrum of Form I is described below and issummarized in FIG. 7.

The Region from 4000 to 400 cm⁻¹

Form I has a large single peak at 3421 cm⁻¹ and also have a broadabsorbance underlying these peaks, from approximately 3300 to 2600 cm⁻¹.There are also two peaks at and 2956 cm⁻¹, likely due to C—H stretch.Form I has peaks at 1791, 1717 and 1671 cm⁻¹. All three forms have apeak at approximately 1560 cm⁻¹

ST-246 Form I is the desired polymorph of ST-246. It appears to be thethermodynamically most stable form, as all other get converted toForm-I.

ST-246 Form I is stable and hence can be stored at ambient conditions.Form I has not been shown to convert to another polymorphic form underseveral environmental and process conditions that a drug couldexperience during various stages of manufacturing and storage. Some ofthe conditions tested include storage at high temperature and highhumidity, room temperature and high humidity, low humidity, up to 60°C., capsule manufacturing using wet granulation and drying, duringmilling or micronization process, in suspension, long term storage atroom temperature. Further, Form-I is non hygroscopic and hence does notabsorb moisture even at 90% relative humidity conditions. Form I isreliably manufactured by the commercial process crystallization processwith more than 99.0% purity and with impurities not more than 0.15%.

Form II

In another aspect of the invention, the crystalline form of ST-246 isdisclosed and is denominated as Form II of the ST-246, or in short “FormII”.

ST-246 Form II has been obtained in the presence of some alcohols, aswell as acetone/IPA mixtures. In the preferred aspect of the invention,Form II is reliably crystallized in the presence of ethyl acetate orchloroform.

Anhydrate Form II is relatively unstable and prone to conversion to FormIII due to absorption of moisture.

Form II is an anhydrate crystalline form of ST-246. Examples of X-RayDiffraction (XRPD) are summarized in FIG. 2.

Form III

In another aspect of the invention, the invention concerns thecrystalline form of ST-246 that is denominated as Form III of theST-246, or in short “Form III”.

As summarized in Tables 1 and 2, IPA/water mixtures, at various waterlevels, tend to give Form III. Further, Form III can be generated from awater slurry of Form V. Based on the data summarized in Table 1, afaster cooling rate and lower isolation temperature may tend to yieldForm III.

Form III is a monohydrate crystalline form of ST-246. Examples of asingle crystal X-Ray Diffraction (XRPD) data for Form III are shown inFIG. 3 and summarized below:

Angle d value Intensity 2-Theta Angstrom Cps Intensity % 6.71 13.15 102100.0 9.05 9.76 5.23 5.1 12.49 7.08 2.77 2.7 13.03 6.79 11.2 11.0 13.796.42 4.61 4.5 14.87 5.95 2.56 2.5 15.72 5.63 3.79 3.7 16.26 5.45 14.814.5 16.74 5.29 30.3 29.7 18.10 4.90 11.4 11.2 18.43 4.81 4.51 4.4 19.944.45 6.46 6.3 21.04 4.22 10.0 9.8 21.51 4.13 9.64 9.4 23.15 3.84 7.287.1 23.51 3.78 4.10 4.0 25.32 3.51 7.28 7.1 26.24 3.39 3.79 3.7 26.873.32 11.2 11.0 27.32 3.26 4.31 4.2 27.72 3.22 3.69 3.6 28.55 3.12 9.128.9 29.08 3.07 5.84 5.7 29.50 3.03 9.84 9.6 29.84 2.99 6.66 6.5 31.272.86 2.46 2.4 33.48 2.67 3.59 3.5 35.36 2.54 3.38 3.3 39.56 2.28 3.493.4

The characteristic infrared spectrum of the Form III is described belowand summarized in FIG. 8.

The Region from 4000 to 2500 cm⁻¹

Form III has a split peak at 3452 and 3397 cm⁻¹. There is also a peak at3008 and 2956 cm⁻¹, likely due to C—H stretch. There are also peaks atfrom approximately 3300 to 2600 cm⁻¹.

The Region from 2000 to 1500 cm⁻¹

Form III has a set of peaks at 1792, 1713 and 1662 cm 1. All of theseare likely due to C═O stretches. There is also a peak at 1560 cm⁻¹,tentatively assigned to N—H deformation.

The Region from 1500 to 400 cm⁻¹

From approximately 1500 to 400 cm⁻¹, there are a variety of lesssignificant peaks.

Form III (monohydrate) can be converted to Form I in competitive slurryexperiments. Conversion from Form I to Form III has never been observed,suggesting that Form I is a more thermodynamically stable Form than FormIII.

However, Form III has an advantage over other less hydrated forms suchas for example Form V in that Form III is fully hydrated and does notabsorb any further amount of moisture under humid storage conditions.

Form IV

Examples of XRPD, single crystal X-ray data for Form IV are shown inFIG. 4.

In a preferred aspect of the invention, Form IV is formed in thepresence of chlorinated solvents and some alcohols such as for example,TFE, 1 butanol, toluene, methylene chloride, chloroform, among others.Anhydrate Form IV is relatively unstable and prone to conversion to FormV, due to absorption of moisture.

Form V

In yet another aspect of the invention, the invention concerns thecrystalline form of ST-246 that is denominated as Form V of the ST-246,or in short “Form V”.

Form V is a hemihydrate crystalline form of ST-246. Examples of XRPDdata for Form V are shown below and summarized in FIG. 5.

Angle d value Intensity 2-Theta ° Angstrom Cps Intensity % 6.39 13.81101 100.0 6.72 13.14 9.56 9.5 8.16 10.82 1.88 1.9 9.04 9.78 3.75 3.79.52 9.28 6.38 6.3 10.52 8.41 4.88 2.1 12.40 7.13 5.06 5.0 12.79 6.927.31 7.3 13.38 6.61 4.13 4.1 14.15 6.25 12.0 11.9 14.57 6.07 11.4 11.415.84 5.59 15.9 15.9 16.32 5.43 10.7 10.6 16.67 5.31 25.7 25.6 17.505.06 21.2 21.1 18.13 4.89 9.19 9.1 18.48 4.80 5.44 5.4 18.78 4.72 16.916.8 19.79 4.48 38.3 38.1 20.68 4.29 17.3 17.2 21.07 4.21 13.9 13.821.54 4.12 5.25 5.2 22.01 4.04 5.81 5.8 22.73 3.91 7.50 7.5 23.60 3.776.38 6.3 25.25 3.52 4.50 4.5 25.73 3.46 20.1 20.0 26.27 3.39 3.94 3.926.73 3.33 5.63 5.6 27.24 3.27 13.3 13.2 29.02 3.07 10.1 10.1 29.50 3.038.06 8.0 29.83 2.99 6.94 6.9 30.44 2.93 9.00 9.0 32.04 2.79 4.50 4.533.52 2.67 7.13 7.1 34.84 2.57 4.69 4.7 35.68 2.51 6.19 6.2 39.78 2.264.31 4.3

The infrared spectrum of the Form V has also been summarized in FIG. 9and is described below. The underlined peaks are considered the mostcharacteristics of the polymorph:

The Region from 4000 to 2500 cm⁻¹

Form V has a split peak at 3464 and 3402 cm⁻¹ along with a second broadsplit peak at 3238 and 3206 cm⁻¹. These peaks are likely due to OH andNH stretches and appear to allow differentiation of the three forms.Form V also has peaks at ˜3008 and 2956 cm⁻¹, likely due to C—H stretch.There are further peaks at approximately 3300 to 2600 cm⁻¹.

The Region from 2000 to 1500 cm⁻¹

Form V has significantly different spectral characteristics in thisregion as compared to other polymorphic forms of ST-246, showing 5 peaksrather than 3, and these are at 1791, 1733, 1721, 1681 and 1667 cm⁻¹.All of these are likely due to C═O stretches. All three forms have apeak at approximately 1560 cm⁻¹, tentatively assigned to N—Hdeformation. Form V has peaks at 1519 and 1497 cm⁻¹.

The region from 1500 to 400 cm⁻¹

From approximately 1500 to 400 cm⁻¹, the infrared spectra of the threeforms show only slight differences, and this region is probably notuseful for differentiating the three forms of ST-246 discussed here.

Form V (hemi-hydrate) was made during early GMP syntheses of ST-246 andis disclosed in WO 2008/130348, WO 2004/112718 and WO 2008/079159. Thedisadvantage of this polymorph is that it is not fully hydrated. Thisform readily absorbs moisture when placed in a humid environment, andhas been shown to convert to Form I in competitive slurry experiments.

Form V tends to form when insufficient water is present to generate themonohydrate form. Form V is formed from ethyl acetate/hexane mixtureswhen the starting ST-246 used for crystallization does not containwater. If the starting ST-246 contains enough water, Form III can beformed. Form V has also been generated by methanol/water and IPA/watermixtures containing low levels of water.

Form VI

In one aspect of the invention, the invention concerns the crystallineform of ST-246 that is denominated as Form VI of the ST-246, or in short“Form VI”.

Form VI is an monohydrate crystalline form of ST-246. Examples of XRPDare summarized in FIG. 6. In the preferred aspect of the invention, FormVI may be formed, as an example, in the presence of nitromethane orchloroform/methanol as crystallization solvents.

In preparing polymorph Forms I, II, III, IV, and V substantially free ofother polymorph forms, crystallization from a mixture of different formsis generally utilized. However, the crystallization technique withregard to producing each of these polymorph forms substantially free ofother polymorph forms is different and described below.

More specifically, the present invention provides isolated Form I thatis at least about 70% pure (i.e. free of other forms), preferably atleast about 80% pure, preferably at least about 90% pure, preferably atleast about 95% pure, more preferably at least about 99% pure, and mostpreferably at least about 99.9% pure.

The present invention provides for isolated Form II which is at leastabout 70% pure (i.e. free of other forms), preferably at least about 80%pure, preferably at least about 90% pure, more preferably at least about99% pure, and most preferably at least about 99.9% pure. Further, thepresent invention provides for isolated Form III which is at least about70% pure, preferably at least about 80% pure, preferably at least about90% pure, preferably at least about 95% pure, more preferably at leastabout 99% pure, and most preferably at least about 99.9% pure.

Also, the present invention provides for isolated Form IV which is atleast about 70% pure (i.e. free of other forms), preferably at leastabout 80% pure, preferably at least about 90% pure, preferably at leastabout 95% pure, more preferably at least about 99% pure, and mostpreferably at least about 99.9% pure.

Further, the present invention provides for isolated Form VI which is atleast about 70% pure (i.e. free of other forms), preferably at leastabout 80% pure, preferably at least about 90% pure, preferably at leastabout 95% pure, more preferably at least about 99% pure, and mostpreferably at least about 99.9% pure.

The crystals, powder aggregates and coarse powder of the polymorphicforms of the ST-246 may be optionally milled and sorted by size afterundergoing conversion. Milling or grinding refers to physically breakingup the large particles or aggregates of particles using methods andapparatus well known in the art for particle size reduction of powders.Resulting particle sizes may range from millimeters to nanometers,yielding i.e. nanocrystals, microcrystals.

The polymorph of the invention may be milled using known millingprocedures such as wet milling to obtain a particle size appropriate fortablet formation and for other formulation types. Finely divided(nanoparticulate) preparations of the polymorph of the invention may beprepared by processes known in the art, for example see internationalpatent application WO 02/00196 (SmithKline Beecham).

In one aspect, the particle size of each of polymorphic Forms I-IV andVI of ST-246 in the present invention has D₉₀ of the volume meandiameter of the particles within the range of about 0.01-200 □m,preferably about 15-50 □m, and most preferably about 0.01-15 □m. Suchparticles are better in chemical and physical stability, good materialflow characteristics, improving the uniformity of dosage forms and thussuitable for bulk preparation and formulation advantages.

Formulations and Administration

Formulations of polymorphic forms of ST-246 may be prepared by processesknown in pharmaceutics art. The following examples (infra) are given toenable those skilled in the art to more clearly understand and topractice the present invention. They should not be considered aslimiting the scope of the invention, but merely as being illustrativeand representative thereof.

The polymorphic salts of the present invention can be administered in avariety of oral and parenteral dosage forms. Oral dosage forms can betablets, coated tablets, hard and soft gelatin capsules, solutions,emulsions, syrups, or suspensions. Parenteral administration includesintravenous, intramuscular, intracutaneous, subcutaneous, intraduodenal,or intraperitoneal administration. Additionally, the salts of thepresent invention can be administered by transdermal (which may includea penetration enhancement agent), buccal, nasal and suppository routes.

For preparing pharmaceutical compositions from the compounds of thepresent invention, pharmaceutically acceptable carriers can be eithersolid or liquid. Solid form preparations include powders, tablets,pills, hard and soft gelatin capsules, cachets, suppositories, anddispersible granules. A solid carrier can be one or more substanceswhich may also act as diluents, flavoring agents, lubricants, suspendingagents, binders, preservatives, tablet disintegrating agents, or anencapsulating material.

In powders, the carrier is a finely divided solid which is in a mixturewith the finely divided active component. In tablets, the activecomponent is mixed with the carrier having the necessary bindingproperties in suitable proportions and compacted in the shape and sizedesired.

Suitable excipients for tablets, coated tablets, and hard gelatincapsules are, for example, microcrystalline cellulose, lactose, cornstarch and derivatives thereof, magnesium carbonate, magnesium stearate,sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth,methylcellulose, sodium carboxymethylcellulose, talc, and fatty acids ortheir salts, e.g., stearic acid. If desired, the tablets or capsules maybe enteric-coated or sustained release formulations. Suitable excipientsfor soft gelatin capsules are, for example, vegetable oils, waxes, fats,semi-solid and liquid polyols. Liquid form preparations includesolutions, suspensions, retention enemas, and emulsions. For parenteralinjection, liquid preparations can be formulated in solution in water orwater/polyethylene glycol solution.

Aqueous solutions suitable for oral use can be prepared by dissolvingthe active component in water and adding suitable colorants, flavors,stabilizing, and thickening agents as desired. Aqueous suspensionssuitable for oral use can be made by dispersing the finely dividedactive component in water with viscous material, such as natural orsynthetic gums, resins, methylcellulose, sodium carboxymethylcellulose,and other well-known suspending agents.

Compositions also may contain, in addition to the active component,colorants, flavors, stabilizers, buffers, artificial and naturalsweeteners, dispersants, thickeners, preservatives, wetting agents,emulsifiers, salts for adjustment of the osmotic pressure, maskingagents, antioxidants and the like.

The compounds of the present invention can be administered intravenouslyin physiological saline solution (e.g., buffered to a pH of about 7.2 to7.5). Conventional buffers such as phosphates, bicarbonates or citratescan be used in the present compositions.

Also included are solid form preparations which are intended to beconverted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions, andemulsions. For preparing suppositories suitable excipients includenatural and hardened oils, waxes, fatty acid glycerides, semi-liquid orliquid polyols. The molten homogenous mixture is then poured intoconvenient sized molds, allowed to cool, and thereby to solidify.Suitable pharmaceutical carriers, excipients and their formulations aredescribed in Remington: The Science and Practice of Pharmacy 1995,edited by E. W. Martin, Mack Publishing Company, 19th edition, Easton,Pa.

The dosage can vary within wide limits and will, of course, be adjustedin each particular case to the individual requirements of the patientand the severity of the condition being treated. A typical preparationwill contain from about 5% to about 95% active compound (w/w). For oraladministration, a daily dosage of between about 0.01 and about 100 mg/kgbody weight per day should be appropriate in monotherapy and/or incombination therapy. A preferred daily dosage is between about 0.1 andabout 300 mg/kg body weight, more preferred 1 and about 100 mg/kg bodyweight and most preferred 1.0 and about 50 mg/kg body weight per day.

Generally, treatment is initiated with smaller dosages which are lessthan the optimum dose of the compound. Thereafter, the dosage isincreased by small increments until the optimum effect under thecircumstance is reached. The daily dosage can be administered as asingle dosage or in divided dosages, typically between 1 and 5 dosagesper day.

The pharmaceutical preparations are preferably in unit dosage forms. Insuch form, the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form.

Appropriate doses will be readily appreciated by those skilled in theart. It will be appreciated that the amount of a polymorph of theinvention required for use in treatment will vary with the nature of thecondition being treated and the age and the condition of the patient andwill be ultimately at the discretion of the attendant physician orveterinarian. The polymorph of the invention may be used in combinationwith other antibacterial drugs such as penicillin, cephalosporin,sulfonamide or erythromycin.

The combinations referred to above may conveniently be presented for usein the form of a pharmaceutical formulation and thus pharmaceuticalformulations comprising a combination as defined above together with apharmaceutically acceptable carrier or excipient comprise a furtheraspect of the invention. The individual components of such combinationsmay be administered either sequentially or simultaneously in separate orcombined pharmaceutical formulations by any convenient route.

When administration is sequential, either the polymorph of the inventionor the second therapeutic agent may be administered first. Whenadministration is simultaneous, the combination may be administeredeither in the same or different pharmaceutical composition.

Using the routes and methods of administration and dosage amountsdescribed hereinabove and the dosage forms described herein below, theindividual polymorph forms, such as Form I, Form II, Form III, Form IV,Form V and Form VI, and mixtures of polymorph forms of the presentinvention can be used for the prevention and treatment of variousdiseases and conditions in humans. By way of example and not oflimitation, in the case of orthopoxvirus infections and associateddiseases, this is accomplished by administering to a patient in need ofsaid treatment who is suffering from orthopoxvirus infections acomposition containing one of the above polymorph forms, such as Form I,Form II, Form III, Form IV, Form V and Form VI, substantially free ofother polymorph forms or mixtures of polymorphs and an inert carrier ordiluent, said composition being administered in an effective amount toprevent or treat said viral infection.

In accordance with this invention, ST-246, either as a polymorph formsubstantially free of other polymorph forms or as a mixture of polymorphforms, is administered in an effective amount to prevent or treatorthopoxviral infection. Any effective amount of such polymorph formsubstantially free of other polymorph forms or mixtures of polymorphforms needed to prevent or treat such viral infection can be utilized inthis composition. In general, in the case oral dosage forms, dosages offrom about 0.5 mg/kg to about 5.0 mg/kg of body weight per day are used.However the amount of such polymorph form, such as Form I, Form II, FormIII, Form IV, Form V and Form VI, substantially free of other polymorphforms or mixtures of polymorph forms in the oral unit dose to beadministered will depend to a large extent on the condition of viralinfection, and the weight of the patient and of course be subject to thephysician's judgment. In one aspect of the invention, Form I is thepreferred ST-246 polymorph form for administration.

In accordance with this invention, the oral unit dosage form containingthe given polymorph form substantially free of other polymorph forms ormixtures of polymorph forms can be preferably administered at a dosageof from about 30 mg to 800 mg per day, more preferably from about 50 mgto about 600 mg per day, administered once to three times during the dayor as needed.

In some aspect of the invention, the polymorph of the present invention,preferably hydrate form of ST-246, may also be used in combination with:(1) a vaccine; (2) Cidofovir, an injectable antiviral medication whichis acyclic nucleoside phosphonate, and is therefore independent ofphosphorylation by viral enzymes, to treat eczema vaccinatum (EV), alife-threatening complication of vaccinia virus infection, and otherrelated disorders; and/or (3) CMX001 (hexadecyloxypropyl-cidofovir), amimic of a naturally occurring lipid, lysolecithin, formed by linking alipid, 3-hexadecyloxy-1-propanol, to the phosphonate group of cidofovir.

The invention also provides pharmaceutical packs or kits comprising oneor more containers filled with one or more crystalline polymorph ofST-246, including Form I, Form II, Form III, Form IV, Form V and FormVI. Optionally associated with such container(s) can be a notice in theform prescribed by a governmental agency regulating the manufacture, useor sale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration. In a certain aspect of the invention, the kit containsmore than one crystalline polymorph of ST-246.

Example 1—Preparation of Polymorphic Form I

More specifically, to prepare ST-246 monohydrate, Form I,cycloheptatriene is reacted with maleic anhydride in the presence oftoluene to yield the major product, endo isomer. The exo isomer isfurther produced from about 7% to 0.6% by crystallization fromtoluene/heptane. Further, hydrazine in the anhydrous or hydrate form isreacted with Methyl 4-(trifluoromethyl) benzoate in the presence ofisopropanol to yield (4-(trifluoromethyl)-benzhydrazide. The product isthen crystallized from isopropanol.

The next step of the synthesis involves condensingendo-tricyclo[3.2.2.0]non-8-endo-6,7-dicarboxylic anhydride and(4-(trifluoromethyl)-benzhydrazide) in isopropanol. The product isisolated by crystallization from isopropanol and the slurry is furtherheated to reflux and held. The resulting solution is cooled and sampledfor reaction completion. After analysis shows reaction completion,carbon and celite are charged and the batch is heated to reflux andheld. After cooling, the batch is filtered to remove these solidmaterials, followed by a filter chase with IPA. The batch is cooled andheld while slurry is formed. The batch is further cooled and held.Contents are centrifuged and the wet cake containing synthesis productis washed with heptane. The wet cake is dried and is referred to aspartially hydrated form of ST-246 (SG3).

The SG3 is charged followed by ethyl acetate. The mixture is heated andheld to ensure dissolution of SG3. A polish filtration is performed onthe batch and an extraneous material check confirms that the filtrationwas successful. Ethyl acetate is used to charge the filter. Afterheating the batch to reflux, Endotoxin reduced (ER) water is charged.The batch is seeded and the final ER water is charged. The batch is heldat reflux and a slurry check is performed.

Further, the batch is cooled, at which time a sample of the slurry isobtained for verification of correct polymorph. The batch is cooledfurther and is held until final isolation on the centrifuge. The finalAPI is dried, milled using a Fitz Mill as described in WO 02/00196. FormI can be prepared by crystallization of ST-246 from a variety ofsolvents and solvent combinations as further summarized in Tables 2 and3:

TABLE 3 Solvent screening study results. Ratio Saturation OverheatGrowth Crystal Solvent A Solvent B (volA:volB) Temp (° C.) Temp (° C.)Temp (° C.) Form 1-Propanol None 30 35 25 II Ethanol Water 1:1 40 45 35II Acetone IPA 4:1, 1:1, 1:4 40 45 35 II ACN Ethyl 1:1 40 45 35 IIacetate Ethanol Toluene 4:1 40 45 35 II Ethanol Water 1:1 50 55 50 IIAcetone IPA 4:1, 1:1, 1:4 50 55 50 II ACN Ethyl 4:1, 1:1 50 55 50 IIacetate Methanol CHCl3 1:4 50 55 50 II Ethanol Toluene 4:1, 1:1, 1:4 5055 50 II Ethanol Water 1:1 30 35 30 II Acetone IPA 4:1, 1:1, 1:4 30 3530 II Water Ethyl 1:1 30 35 30 II acetate DMF ACN 4:1 30 35 30 IIEthanol Toluene 4:1 30 35 30 II Ethyl acetate None 1 gram scale II IPANone 30 35 25 III DMF None 30 35 25 III DMA None 30 35 25 III PyridineNone 30 35 25 III Isopropyl ether None 30 35 25 III THF None 30 35 25III CH2Cl2 IPA 4:1, 1:1, 1:4 40 45 35 III Ethanol Water 4:1, 1:4 40 4535 III ACN Ethyl 4:1 40 45 35 III acetate TFE THF 4:1, 1:1, 1:4 40 45 35III DMF ACN 4:1, 1:1 40 45 35 III Methanol CHCl3 4:1, 1:1 40 45 35 IIIEthanol Toluene 1:1 40 45 35 III CH2Cl2 IPA 4:1, 1:1, 1:4 50 55 50 IIIEthanol Water  4:01 50 55 50 III ACN Ethyl 1:4 50 55 50 III acetate TFETHF 4:1, 1:1 50 55 50 III DMF ACN 4:1, 1:1, 1:4 50 55 50 III MethanolCHCl3 4:1, 1:1 50 55 50 III Water IPA 1:1, 1:4 30 35 30 III EthanolWater 4:1 30 35 30 III Water Ethyl 4:1 30 35 30 III acetateTrifluoroethanol THF 4:1 30 35 30 III (TFE) DMF ACN 1:1, 1:4 30 35 30III Methanol CHCl3 4:1, 1:1, 1:4 30 35 30 III Ethanol Toluene 1:1, 1:430 35 30 III Trifluoroethanol None 30 35 25 IV (TFE) 1 Butanol None 3035 25 IV CH2Cl2 None 30 35 25 IV CHCl3 None 30 35 25 IV Toluene None 3035 25 IV ACN Ethyl 1:4 40 45 35 IV acetate Ethanol Toluene 1:4 40 45 35IV Water Ethyl 1:4 30 35 30 IV acetate Trifluoroethanol THF 1:1 30 35 30IV (TFE) 1 Butanol None 1 gram scale IV Methanol None 30 35 25 V EthanolNone 30 35 25 V 2 Butanol None 30 35 25 V Acetone None 30 35 25 V MethylEthyl None 30 35 25 V Ketone Ethyl acetate None 30 35 25 V MTBE None 3035 25 V Isopropyl acetate None 30 35 25 V Acetonitrile (can) None 30 3525 V DMF ACN 1:4 40 45 35 V Nitromethane None 30 35 25 VI NitromethaneNone 1 gram scale VI Methanol CHCl3 1:4 40 45 35 VI

The identity of ST-246 Form I obtained by the above described processwas confirmed by XRPD and IR as summarized in FIGS. 1 and 7.

Example 2—Preparation of Polymorphic Form II

Standards of Form II were made by re-crystallization of Forms I and Vstarting material from ethyl acetate and chloroform solvents. An amountof ST-246 Form I or Form V was dissolved in either ethyl acetate orchloroform and filtered through 0.45 □m membrane filters. The filteredsolutions were overheated at a higher temperature to make sure allsolids were dissolved and then reduced to a lower temperature andevaporated to dryness under a nitrogen purge (˜2 psi.).

The preferred crystallization conditions for Form II are summarized inTable 4 below:

TABLE 4 Crystallization conditions for ST-246 Form II. EvaporationStarting Overheating Temperature XRPD Material Solvent Temperature (°C.) (° C.) Pattern 1.5 g Ethyl acetate 35 25 Form II 1.1 g Chloroform 4535 Form II

The further examples of crystallization conditions for Form II are alsosummarized in Tables 1-2 above.

The identity of ST-246 Form II obtained by the above described processwas confirmed by XRPD as summarized in FIG. 2.

Example 3—Preparation of Polymorphic Form III

Form III is produced from reslurry of anhydrous ST-246 in water. Furtherexamples of solvents used to generate Form III are summarized in Tables1-9 above.

The identity of ST-246 Form III produced by the process described abovewas confirmed by XRPD and IR as summarized in FIGS. 3 and 8.

Example 4—Preparation of Polymorphic Form IV

A standard of Form IV was made by re-crystallization of Form I startingmaterial from 1-butanol solvent. The starting solid material wasdissolved in 1-butanol and filtered through a 0.45 um membrane filter.The filtered solution was overheated at a higher temperature to makesure all solids were dissolved and then reduced to a lower temperatureand evaporated to dryness under a nitrogen purge (˜2 psi). The preferredcrystallization conditions for Form IV are summarized in Table 5.Further examples of crystallization of Form IV are summarized in Tables1-3.

TABLE 5 Crystallization conditions for ST-246 Form IV. EvaporationStarting Overheating Temperature XRPD Material Solvent Temperature (°C.) (° C.) Pattern 1.5 g 1-butanol 50 35 Form IV 70 ml

The identity of ST-246 Form IV produced by the process described abovewas confirmed by XRPD as summarized in FIG. 4.

Example 5—Preparation of Polymorphic Form V

Form V (hemi-hydrate) was made during early GMP syntheses of ST-246 andis disclosed in WO 2008/079159 and WO 2008/130348. The disadvantage ofthis polymorph is that it is not fully hydrated. This form readilyabsorbs moisture when placed in a humid environment, and has been shownto convert to Form I in competitive slurry experiments

The identity of ST-246 Form V was confirmed by XRPD and IR as summarizedin FIGS. 5 and 9.

Example 6—Preparation of Polymorphic Form VI

A standard of Form VI was made by re-crystallization of Form V startingmaterial from nitromethane solvent. The starting solid material wasdissolved in nitromethane and filtered through a 0.45 um membranefilter. The filtered solution was overheated at a higher temperature tomake sure all solids were dissolved and then reduced to a lowertemperature and evaporated to dryness under a nitrogen purge (˜2 psi).Form VI can be prepared by crystallization of ST-246 from a variety ofsolvents and solvent combinations. The preferred crystalizationconditions are summarized in Table 6 below. Further examples ofcrystallization of Form VI are summarized in Tables 1-3.

TABLE 6 Form VI Crystallization Conditions. Evaporation StartingOverheating Temperature XRPD Material Solvent Temperature (° C.) (° C.)Pattern 1.2 g Nitromethane 35 25 then 35 Form VI (60 ml)

The identity of ST-246 Form VI obtained by a process described above wasconfirmed by XRPD as summarized in FIG. 6.

Example 7—Distinguishing and Comparative Physical Characteristic ofPolymorphic Forms of ST-246, Form I, Form III and Form V

It has been determined that ST-246 can exist in three predominantphysical forms (Forms I, III and V). The data was obtained on therelevant physical/chemical properties and stability of the polymorphs todetermine if the different solid forms impact the quality of theproduct. These data include the crystallographic properties of thepolymorphs and physical/chemical properties of the polymorphs (e.g.solubility, dissolution, melting range) accelerated stability data.

The X-ray diffractions patterns of Forms I, III and V are shown in FIGS.1, 3 and 5, respectively. The powder X-ray pattern of Forms I, III, andV are readily distinguishable based on the unique features in theirpowder patterns.

Interconversion of Forms I, III and V

Competitive and non-competitive slurry experiments were performed todetermine the most stable form of ST-246. The slurry experiments wereperformed by exposing excess material of one or more forms of ST-246 ina small volume of water and agitating the resulting suspensions forseveral days at ambient temperature and/or 45° C. Similar experimentswere also done at different pH values for 60 minutes at 37° C. Theslurry was filtered and the solid analyzed by powder XRPD. To avoidpossible desolvation or physical change after isolation, the sampleswere not subjected to drying before powder X-ray analysis. Competitiveand non-competitive slurry experiments in water show that Form V andForm III convert to Form I in water and Form I remains unchanged. Theslurry data are summarized in Table 7 below:

TABLE 7 Interconversion of Polymorphs of ST-246 in Aqueous Liquids.Initial Forms Solvent/Temp Slurry Duration Final Form I, III & VWater/RT  5 days I I, III & V Water/RT 13 days I I & III Water/RT  2days I I &III Water/RT 30 days I V pH 1.2, 6.8/37° C. 30 min III IIIWater/45° C. 17 days I I Water/45° C. 17 days IMicronization of Form I and III:

ST-246 belongs to BCS class II due to its poor solubility inphysiologically relevant buffers. Phase 1 clinical trial material wasmade using micronized Form V with particle size of d50% 4.8 μm and d90%12 μm. Hence both Forms I and III at a scale of 400 μm were micronizedusing an airjet mill as described in WO 02/00196. On milling, both theforms yielded the desired particle size without undergoing anytransformation in physical form (based on XRPD).

Representative XRPD patterns of both micronized and unmicronized Forms Iand III are summarized in FIGS. 14 and 15.

Drug Substance Stability

Drug substance Forms I and III, both micronized and unmicronized, haveundergone short-term stability evaluation under stress conditions. Theshort-term study has been completed and the data obtained at 40° C./75%RH are presented in Tables 8 and 9.

TABLE 8 Three months R&D stability data of ST-246 Forms I and III. 40°C./75% RH Open Test Initial 1 Month 2 Month 3 Month Batch #14KM46B (FormI) Assay (HPLC) 97.38% Not Done Not Done 98.53% Related Substances 0.06%isomer, Not Done Not Done 0.07% isomer, <0.05% unknown <0.05% unknownRRT = 1.4 RRT = 1.4 Moisture (by KF) 4.7% 5.1% 5.0% 5.2% XRD Form I FormI Form I Form I Melting Point (by 197.15° C. 196.39° C. 196.21° C. Notdone DSC) Batch #14KM49B (Form III) Assay (HPLC) 100.64% Not Done NotDone 99.95% Related Substances 0.02% isomer, Not Done Not Done 0.03%isomer, <0.05% unknown <0.05% unknown RRT = 1.4 RRT = 1.4 Moisture (byKF) 4.5% 5.2% 5.1% 4.9% XRD Form III Form III Form III Form III MeltingPoint (by 196.92° C. 195.85° C. 196.24° C. Not done DSC)

TABLE 9 R&D Stability Data of ST-246 Forms I and III (Micronized lot).25° C./60% RH 40° C./75% RH Test T = 0 1 Month 2 Month 1 Month 2 MonthLot # 14KM75B-4724 (Form I) Assay (HPLC) % 97.36 101.15 99.68 102.3399.71 Related Substances (%) RRT 1.08 0.11 0.07 <0.05 0.06 0.08 RRT 1.37<0.05 <0.05 <0.05 <0.05 <0.05 RRT 1.39 <0.05 <0.05 <0.05 <0.05 <0.05Moisture (by TGA) % 4.8 4.7 5.09 4.7 5.26 XRD Form I Form I Form I FormI Form I Melting Point (by DSC) 196.33° C. Not Done 196.03° C. Not Done196.06° C. Lot # 14KM84-4724 (Form III) Assay (HPLC) % 97.87 101.6 99.48102.1 99.38 Related Substances (%) RRT 1.08 0.07 0.10 <0.05 0.07 0.05RRT 1.37 <0.05 <0.05 <0.05 <0.71 <0.05 RRT 1.39 <0.05 <0.05 <0.05 <0.09<0.05 Moisture (by TGA) % 4.8 4.7 4.78 5.2 5.32 XRD Form III Form IIIForm III Form III Form III Melting Point (by DSC) 194.44° C. Not DoneNot Done Not Done Not Done

The data on both micronized and unmicronized drug substance indicates nochange in physical form of both I and III with respect to Purity,Related Substances, Moisture, XRPD and DSC.

The long-term study has also been completed and the data obtained arepresented in Table 10.

TABLE 10 Long-Term Stability Testing Results - Lot # SG-08B01-M(Form-I). HPLC Related SG1 & Interval Water HPLC Substances SG1 ExoHydrazine month Description Content Assay RRt~1.08 Total Isomer By HPLCSG2 Dimer 0 conforms 4.40% 99.7% 0.06% 0.06% na na na 3 conforms 4.66%99.7% 0.06% 0.06% na na na 6 conforms 4.71% 99.7% <0.05% <0.05% na na na9 conforms 4.47% 97.4% <0.05% <0.05% na na na 12 conforms 4.67% 98.5%<0.05% <0.05% na na na 18 conforms 4.79% 99.8% 0.05% 0.05% na na na 24conforms 4.81% 100.2% 0.05% 0.05% <0.05% <0.1 ppm <0.01%Static Sorption of Forms I, III and V

Hygroscopicity testing was done on Forms I, III and V at varioushumidity conditions to understand sorption/desorption properties.Approximately 1 g of each form was ground with a mortar and pestle.Water content was determined by TGA. Approximately 100 mg of each powderwas placed in static humidity chambers at 11 and 97% RH at approximately25° C. for 10 days. The only sample that exhibited a change in weightloss from Day 0 was the Form V sample stored at 97% RH. The data issummarized in Table 11 below:

TABLE 11 Hygroscopicity data of Forms I, III and V at 11% RH and 97% RH.Sample % RH Day 12 wt loss Day 0 wt loss Form I 11 4.8% to 117.5° C.4.8% to 117° C. Form III 11 4.8% to 97.5° C.  4.8% 97.3° C. Form V 112.2% to 109.8° C.  2.2% to 97.8° C. Form I 97.6 4.7% to 119° C.   4.8%to 117° C. Form III 97.6 4.8% to 100.2° C. 4.8% 97.3° C. Form V 97.63.4% to 112.2° C. 2.2% to 111° C.

Example 8.—Analysis of the Effect of ST-246 API Particle Size onDissolution Profiles of ST-246 Hard Gelatin Capsules

The effect of the particle size of drugs on their dissolution profilehas been extensively reviewed (see Fincher et al., 1968) and it had beenhypothesized that a decrease in particle size of sparingly soluble drugsresults in increased dissolution rates owing to the increased surfacearea of the drug exposed to the solvent.

Table 12 summarizes micronized and unmicronized ST-API particle size inmicrons, wherein micronization done for further testing the effect ofparticle size on the dissolution profiles of ST-246.

TABLE 12 Micronized and unmicronized ST-246 API particle size inmicrons. Particle size of ST-246 API in microns ST-246 API Lot# D10 D50D90 Micronized SG-08G02-M 1.008 3.243 8.097 SG-08H05-M 0.918 2.517 5.987SG-08H06-M 1.007 2.602 5.627 SG-08K07-M 0.909 2.450 5.563 SG-08L08-M1.032 2.479 4.999 SG-08-09-M-Trial 1 1.233 2.166 4.617 SG-08-09-M-Trial2 1.587 4.802 13.601 SG-08-09-M-Trial 3 1.723 5.846 17.698SG-08-09-M-Trial 4 1.888 7.111 22.806 Un micronized SG-08-09-Munmicronized 17.4 111.8 281.2

In order to evaluate the effect of particle of API on dissolution ofST-246 capsules, the following formulation comprising ST-246 polymorphForm I was evaluated. For these experiments, ST-246 (Form-I), 200 mgcapsules were prepared using drug substance with different particle sizedistributions, such as d90% less than 10 μm, d90% 16 um, d90% 25 um,d90% less than 254 μm and d90% less than 75 μm. The composition of theST-246 gelatin capsules are shown in the Table 13 below.

TABLE 13 Composition of capsules used for discriminating dissolutionmedium experiments. Lot# SJI091023- SJI091023- SG-09K10-Q- SG-09K10-Q-Lot# WW386- Lot# DN401- 0801637 API-Trial #3 API-Trial #4 API-40 umAPI-60 um 89 93 mg/Capsule mg/Capsule mg/Capsule mg/Capsule mg/Capsulemg/Capsule mg/Capsule Particle size distribution [d90% [d90% [d90% d90%d90% [d90% [d90% ST-246 Composition Ingredient 5.3 μm] 16.6 μm] 26.6 μm]40.85 μm] 58.20 μm] 75 μm] 254 μm] ST-246 (Form-I) Monohydrate 200 200200 200 200 200 200 Microcrystalline cellulose, NF 88.60 88.60 88.6088.60 88.60 88.60 88.60 Lactose monohydrate, NF 33.15 33.15 33.15 33.1533.15 33.15 33.15 Croscarmellose sodium, NF 42.90 42.90 42.90 42.9042.90 42.90 42.90 Colloidal silicon dioxide, NF 1.95 1.95 1.95 1.95 1.951.95 1.95 Hydroxypropyl methylcellulose, USP 13.65 13.65 13.65 13.6513.65 13.65 13.65 Sodium lauryl sulfate, NF 7.80 7.80 7.80 7.80 7.807.80 7.80 Magnesium stearate NF 1.95 1.95 1.95 1.95 1.95 1.95 1.95Capsule weight 390 390 390 390 390 390 390For these experiments, ST-246 (Form I) dissolution profiles aredetermined in an USP apparatus 2 (paddle) which runs at rpm. Thedissolution profiles are determined at 37° C., in a 900 ml dissolutionmedium, containing 0.05 M Phosphate buffer pH 7.5, containing 3% HDTMA.Cumulative drug release over time is represented as a percent of ST-246%dissolved and is plotted as a function of dissolution medium samplingtime.

As summarized in Table 14 and FIG. 16-20, ST-246 (Form I) with a D90particle size (d90%) of 5.3 microns and 16.6 microns achieved almost100% dissolution at approximately 22 minutes, whereas ST-246 (Form I)with a D90 particle size (d90%) of 26.6 achieved almost 100% dissolutionat 30 minutes. Also, ST-246 (Form I) with a D90 particle size of 40.85and 58.2 microns achieves almost 85 to 86% dissolution in 30 minutes.Further, ST-246 (Form I) with a D90 particle size of 75 microns achievesalmost 86% dissolution in 30 minutes and ST-246 (Form I) with D90particle size of 254 microns achieves only 44% dissolution in 60minutes. Table 14 shows dissolution profiles using an alternatedissolution method (1% HDTMA in 900 mL of 0.05 M Phosphate buffer pH 7.5at 37° C. in an USP apparatus 2 (paddle) which runs at 50 rpm) for thecapsules made with ST-246 (Form I) with a D90 particle size (d90%) of5.3 microns, 16.6 microns, and 26.6 microns.

TABLE 14 The % Dissolution of ST-246 (Form I) Capsules with API ofVarious Particle Sizes in 3% HDTMA in 900 ml of 0.05M Phosphate buffer,pH 7.5, using dissolution apparatus USP 2 with a paddle speed of 75 RPMat 37° C. % Dissolution SG-09K10- SG-09K10- Lot# SJI091023- SJI091023-Q-API- Q-API- Lot# Lot# 0801637 API-Trial #3 API-Trial #4 40 um 60 umWW386-89 DN401-93 Time in d90% d90% d90% d90% d90% d90% d90% Minutes 5.3μm 16.6 μm 26.6 μm 40.85 μm 58.20 μm 75 μm 254 μm 15 88 85 76 70 67 6922 30 98 96 91 82 80 81 31 45 99 98 95 86 85 87 39 60 101 99 97 89 88 9444 % RSD SG-09K10- SG-09K10- Lot# SJI091023- SJI091023- Q-API- Q-API-Lot# Lot# 0801637 API-Trial #3 API-Trial #4 40 um 60 um WW386-89DN401-93 Time in d90% d90% d90% d90% d90% d90% d90% Minutes 5.3 μm 16.6μm 26.6 μm 40.85 μm 58.20 μm 75 μm 254 μm 15 5 5 13 2.8 4.8 3 2 30 4 1 42.6 3.9 5 3 45 3 1 3 2.5 2.8 4 3 60 2 2 2 2.5 3.2 3 3

TABLE 15 The % Dissolution of ST-246 (Form I) Capsules with API ofVarious Particle Sizes in 1% HDTMA in 900 ml of 0.05M Phosphate buffer,pH 7.5, using dissolution apparatus USP 2 with a paddle speed of 50 RPMat 37° C. % Dissolution % RSD Lot# SJI091023- SJI091023- Lot# SJI091023-SJI091023- 0801637 API-Trial #3 API-Trial #4 0801637 API-Trial #3API-Trial #4 Time in d 90% d 90% d 90% d90% d 90% d 90% Minutes 5.3 μm16.6 μm 26.6 μm 5.3 μm 16.6 μm 26.6 μm 15 44 55 56 22 12 11 30 75 70 7111 9 7 45 87 77 77 3 8 7 60 94 81 81 3 8 7

Further, ST-246, Form I, can be formulated for oral administration incapsules comprising 200 mg of ST-246. For these experiments, ST-246(Form I) with a D90 particle size of between about 5.3 to 75 microns maybe used. All inactive ingredients may be GRAS and USPLNF excipients. Themanufacturing process may include wet granulation using a high shearmixer/granulator and filling into hard gelatin capsules.

Suitable dosage forms can include capsules containing various amounts ofactive ingredient. The quantitative composition of exemplary dosage formcontaining 200 mg of ST-246 monohydrate, micronized with a D90 particlesize of less than about 10 microns, is summarized in Table below:

TABLE 16 Quantitative Composition of ST-246 Drug Product 200 mg StrengthIngredient Function mg/Capsule % w/w ST-246 monohydrate^(a) (micronized,D₉₀ < 10 microns) Active Ingredient; white to 200.00 51.28 monohydrate,based on anhydrous basis. off-white powder Microcrystalline cellulose,NF^(b) Water Insoluble Diluent 88.60 22.72 Lactose monohydrate, NF Watersoluble Diluent 33.15 8.50 Croscarmellose sodium, NF^(b) Disintegrant42.90 11.00 Colloidal silicon dioxide, NF Glidant 1.95 0.50Hypromellose, USP Binder 13.65 3.50 Sodium lauryl sulfate, NF WettingAgent/Solubilizer 7.80 2.00 Magnesium stearate NF Lubricant 1.95 0.50Water USP^(c) Granulating Agent Hard Gelatin Capsule shell,orange/black, Size 0 Encapsulation 1 capsule — Capsule weight, mgs 390100 ^(a)The quantity of ST-246 monohydrate may be adjusted based on thedrug substance assay, which is calculated to reflect the purity andwater content. The amount of Lactose will be adjusted to maintain thesame capsule weight. ^(b)Microcrystalline cellulose and croscarmellosesodium are added as intra granular and extra granular excipients.^(c)Removed during processing.

Other examples of compositions are summarized in Table 17.

TABLE 17 Quantitative Composition of ST-246 Drug Product 200 mg StrengthIngredient Function mg/Capsule ST-246 monohydrate^(a) (micronized, D₉₀ <10 microns) Active Ingredient; white to 200.00 monohydrate, based onanhydrous basis. off-white powder Microcrystalline cellulose, NF^(b)Water Insoluble Diluent 88.60 Lactose monohydrate, NF Water solubleDiluent 40.95 Croscarmellose sodium, NF^(b) Disintegrant 42.90 Colloidalsilicon dioxide, NF Glidant 1.95 Hypromellose, USP Binder 13.65Magnesium stearate NF Lubricant 1.95 Water USP^(c) Granulating AgentHard Gelatin Capsule shell, orange/black, Size 0 Encapsulation 1 capsuleCapsule weight, mgs 390

Example 9—Inhibition of Orthopox Viral Replication

The ability of the Form I of ST-246 to inhibit Vaccinia virus isestablished by the following experimental procedure:

Preparation of Virus Stock

Virus stocks of Vaccinia virus (NYCBH) are prepared in Vero cellsinfected at low multiplicity (0.01 plaque forming units (PFU)/cell) andharvested when cytopathic effects were complete (4+CPE). The samples arefrozen and thawed and then sonicated to release cell-associated virus.The cell debris are removed by low-speed centrifugation, and theresulting virus suspension is stored in 1 mL aliquots at −80.degree. C.The PFU/mL of the virus suspension is quantified by standard plaqueassay on Vero and BSC-40 cells.

Vaccinia CPE Assay

To determine the amount of vaccinia virus stock required to producecomplete CPE in 3 days, Vero cell monolayers are seeded on to 96-wellplates and infected with 2-fold serial dilutions of the vaccinia virusstock. At 3 days post-infection, the cultures are fixed with 5%glutaraldehyde and stained with 0.1% crystal violet. Virus-induced CPEis quantified spectrophotometrically at OD.sub.570. From this analysis,a 1:800 dilution of vaccinia virus stock is chosen for use in the HTSassay. This amount of vaccinia virus represents a multiplicity ofinfection of approximately 0.1 PFU/cell.

To establish the signal-to-noise ratio (S/N) of the 96-well assay andevaluate the well-to-well and assay-to-assay variability, sixindependent experiments are performed. Vero cell monolayers are infectedwith 1:800 dilution of vaccinia virus stock.

Each plate contains the following controls: quadruplicate virus-infectedwells, quadruplicate uninfected cell wells and a dose response curve induplicate for cidofovir (CDV) added at 300, 100, 30 and 10 DAM, orphosphonoacetic acid (PAA) added at 2100, 714, 210, and 71 M asreference standards. At day 3 post-infection, the plates are processedas described above. The results of these experiments indicate that the96-well assay format is robust and reproducible.

Form I Testing

ST-246, Form I is tested in the vaccinia virus CPE assay. Form I isdissolved in DMSO and diluted in medium such that the finalconcentration in each well is 5 pM compound and 0.5% DMSO. Form I isadded robotically to the culture medium using the Biomek® FX robotsystem.

Following compound addition, the cultures are infected with vacciniavirus. After 3 days, plates are processed and CPE quantified asdescribed. ST-246 Form I of the invention inhibited vacciniavirus-induced CPE by greater than 50% at the test concentration(5.mu·M). Form I is further evaluated for potency (EC₅₀) in the CPEassay and cytotoxicity (CC.sub.50) in an MTT assay. The MTT assaymeasures mitochondrial dehydrogenase activity in dividing cells. Theabsorbance of the formazin at 490 nm can be measured directly from96-well assay plates following solubilization of the formazin in 50%ethanol. The quantity of formazin product is directly proportional tothe number of living cells in culture. The EC₅₀ values are determined bycomparing compound-treated and compound-untreated cells using a computerprogram. Thus, the EC₅₀ value of ST-246 Form I in the CPE assay is 50nM.

The specificity of ST-246, Form I, for orthopox virus inhibition isreflected in the fact that they do not inhibit the replication ofunrelated viruses, including Pichinde virus, Rift Valley fever virus(strain MP12), respiratory syncytial virus and cytomegalovirus.

Example 10—ST-246, Form I In Vivo Studies

Study Design

The study is designed as a randomized, placebo-controlled,parallel-group, longitudinal study of oral ST-246, Form I, in cynomolgusmonkeys (Macaca fascicularis). For these experiments, 15 NHPs areinfected with 5×10⁷ PFU of the Zaire 79 strain of MPX by i.v. injectionand are randomized into five treatment arms of three NHP each. Thevehicle or ST-246, Form I, are administered at 3 mg/kg, 10 mg/kg, 30mg/kg, and 100 mg/kg orally once per day followed by 5 ml/kg of a 30%suspension of hydrated homogenized monkey biscuits.

Treatment starts on day 3 post-infection and has continued once dailyfor 14 days. The infected animals are observed at least twice each dayfor up to 33 days to examine them for signs of illness. Blood samplesare collected from the infected animals for virological, hematological,immunological, and chemical analyses. A full necropsy is performed onanimals that died during the study to collect tissues for pathologicalexamination. To determine the extent of infection, three animals areeuthanized on day 3 and their tissues are processed to determine thevirus levels in tissues. The organs are freeze-thawed, and a 10% tissuehomogenate is produced and analyzed by quantitative PCR. The number ofMPX genomes per milliliter of blood is determined by the extraction ofDNA with a Qiagen QIAmp DNA minikit and quantitative TaqMan-MGB PCR, asdescribed previously. Monkeypox lesions are enumerated daily.

Pharmacokinetic Analysis of ST-246, Form I, in Cynomolgus Monkeys (NHP).

The NHP are administered ST-246 at 10, 20, and 30 mg/kg by oral gavageunder fed conditions. On the dosing days, all animals are administered aprimate biscuit slurry immediately prior to dose administration. Thebiscuit slurry contained one can (8 oz) of Ensure or other liquid diet,approximately 36 g of fluid and electrolyte replacement formula (Prang),approximately 94 g of infant formula, 1 jar (2.5 oz) of strained fruit,five monkey chow biscuits, and approximately 2 oz of water blendedtogether to achieve a uniform consistency. The biscuit slurry isadministered via oral gavage at a dose volume of 10 ml/kg. After eachdose of the test article is administered and prior to the removal of thegavage tube, the tube is flushed with 10 ml of tap water. The dosingformulations are stirred with a magnetic stir bar and stir plate priorto and throughout the administration. Individual doses are based on themost recent body weights. With the exception of the 20-mg/kg dose group,each dose group contains three male and three female NHP; the 20-mg/kgdose group contained four male and four female NHP. The NHP in the 10-and 30-mg/kg dose groups received ST-246, Form I, for 14 consecutivedays, and those in the 20-mg/kg dose group received a single dose ofST-246. Blood samples are collected from the femoral artery/veinpretesting (0 h) (prior to dosing) and at 0.5, 1, 2, 3, 4, 6, 8, 12, and24 h postdosing on days 1, 7, and for determination of the plasmaconcentrations of ST-246, Form I.

Evaluation of ST-246, Form I, Efficacy in Cynomolgus Monkey Model ofMonkeypox.

The study described here is randomized, placebo-controlled,parallel-group, longitudinal study of oral ST-246, Form I, in NHPinfected i.v. with MPX. The animals exhibit extensive MPX infection ofmajor tissues, as demonstrated by the level of virus in tissues' At thetime of ST-246, Form I, treatment, one-third of the NHP has virallesions. All animals receiving vehicle alone either die or requireeuthanasia because they are moribund during the study period of 33 days,while all animals receiving ST-246, Form I, survive. Each of the animalsreceiving ST-246, Form I, survive the full study.

For these experiments, viral load and lesion development are quantifieddaily. All doses of ST-246, Form I, significantly decrease the amount ofviral DNA present compared to that in the vehicle-treated animalsbeginning on dpi. At the end of the 14-day treatment, the viral loads inthe ST-246, Form I-treated groups show a linear dose-response, with alltreatment groups showing a significant reduction in the level of virusreplication of more than 1,000-fold compared to that in thevehicle-treated group.

Example 11—Pharmacokinetic Comparison of a Single Oral Dose of Form IVersus Form V Capsules of the Anti-Orthopoxvirus Compound ST-246 inHealthy Human Volunteers

Study Design

This was a Phase I, randomized, double-blind, crossover, exploratorystudy to compare the pharmacokinetics (PK; AUC variables and C_(max)) ofa single 400 mg (2×200 mg) oral dose of ST-246 Form I (the test) withST-246 Form V (the reference), and to evaluate the safety andtolerability of both Forms in fed normal healthy volunteers. Twelve ofsixty-three screened individuals (males and non-pregnant females, 18 to50 years old inclusive) were accepted into the study, and wererandomized to one of the following sequences: Form I then Form V, orForm V then Form I.

To determine the PK of ST-246, a urine and baseline (0 hour) venousblood sample were obtained on Day 1, followed by serial blood drawsafter medication administration. All subjects received a single, 400-mgdose (2×200 mg) of either Form I or Form V of ST-246, orallyadministered within 30 minutes after a standard light meal. Post-dose(Treatment 1) blood samples for PK analyses were taken at 0.5, 1, 2, 3,4, 8, 12, 24, 36, 48, and 72 hours. A post-dose urine sample wasobtained on Day 2. A Washout Period occurred during study Days 2-10, soTreatment 2 occurred on Day 11. At this time, those subjects originallyreceiving Form I of ST-246, now received a single, 400 mg dose (2×200mg) of Form V, and vice versa. Blood sampling for PK analyses followingTreatment, 2, occurred as for post-Treatment 1, and urine samplingoccurred on Day 14. Plasma samples were collected and stored at −70° C.until analyzed for maximum drug concentration [Cmax], time to maximumdrug concentration [Tmax], terminal half-life [t½], area under theconcentration-time curve [AUC], and renal clearance [Clr]. Urine sampleswere immediately centrifuged at 4° C. for 10 min at 2,000×g, andevaluated for urinary excretion. ST-246 was quantified from human plasmaspecimens by a validated liquid chromatography and tandem massspectrometry method using an analog of ST-246 as an internal standard.

The study compared the pharmacokinetic (PK) profiles of ST-246 Form 1and Form V capsules following a single oral dose administration. Thisobjective was achieved through the collection and analysis of plasmasamples for PK assessment of Form I from 12 of 12 subjects and of Form Vfrom 11 of 12 subjects. Pharmacokinetic parameters, peak plasmaconcentration (Cmax,), time at which Cmax is attained post doseadministration (Tmax), plasma exposure (AUC0-τ, AUC0-∞) and eliminationhalf-life (t½) were estimated for ST-246 by applying non-compartmentalanalysis using WinNonlin professional edition software (PharsightCorporation, Version 5.2).

The pharmacokinetic parameters for ST-246, Form I and Form V, aresummarized in Table 18 below:

TABLE 18 Pharmacokinetic parameters for ST-246, Form I and Form V. FormAUC_(0-τ) AUC_(0-∞) AUC_((extrap)) t_(1/2) C_(max) T_(max) GroupStatistics (hr*ng/mL) (hr*ng/mL) (%) (hr) (ng/mL) (hr) Form I N 12 11 1111 12 12 Mean 15624.5 19922.02 17.444 27.446 1068.9 3.8 SD 5449.1886543.563 7.84 13.109 294.3 1.5 CV % 34.876 32.846 44.947 47.763 27.539.6 Geometric 14816.26 19049.63 15.748 24.746 1026.9 3.5 Mean Median14151.15 17201.75 13.214 25.12 1170 3.5 Minimum 8053.5 13959.18 5.710.94 525 2 Maximum 26596.58 31058.8 30.4 56.48 1590 8 Missing 0 1 1 1 00 Form V N 11 8 8 8 11 11 Mean 20065.32 21982.71 15.275 29.18 1230.2 3.8SD 6744.974 9330.953 10.811 21.992 348.6 1.6 CV % 33.615 42.447 70.7875.365 28.3 41.9 Geometric 19020.83 20409.17 12.369 23.083 1185 3.6 MeanMedian 19398.5 19465.47 12.465 16.647 1180 4 Minimum 10398.53 11946.954.53 11.48 732 2 Maximum 30974 39058.28 37.51 69.45 1940 8 Missing 0 3 33 0 0 NOTE: For a given variable and drug form, geometric mean was notcalculated if any of the values were 0 KEY: AUC_(0-∞) = Area under theplasma concentration-time curve from time zero to infinity; AUC_(0-τ) =Area under the drug concentration-time curve from time zero to time twhere t is the last time-point with a drug concentration ≥ lowestobtainable quantification; AUC_((extrap)) = Area under the curveextrapolated; t_(1/2) = Terminal half-life; C_(max) = maximum plasmaconcentration; CV % = Coefficient of variance; h = hours; N = Number ofsubjects; PK = Pharmacokinetics; SD = Standard deviation; T_(max) = Timeto maximum plasma concentration

The mean (SD) ST-246 plasma concentrations over time (PK population) areshown in FIG. 24 after a single oral administration.

What is claimed is:
 1. A polymorph Form VI of4-trifluoromethyl-N-(3,3a,4,4a,5,5a,6,6a-octahydro-1,3-dioxo-4,6-ethenocycloprop[f]isoindol-2(1H)-yl)-benzamide(ST-246) which shows an X-ray powder diffraction pattern havingcharacteristic peaks at a reflection angle 2θ of 6.43, 7.10, 8.23,10.46, 12.62, 13.72, 15.71, 17.84, 19.34, 21.52, 23.73, 24.17, 24.71,26.71, 27.26, 30.11 and 31.00 degrees.
 2. A polymorph according to claim1 that is at least about 70% free of other forms.
 3. A polymorphaccording to claim 1 that is at least about 80% free of other forms. 4.A polymorph according to claim 1 that is at least about 90% free ofother forms.
 5. A polymorph according to claim 1 that is at least about95% free of other forms.
 6. A polymorph according to claim 1 that is atleast about 99% free of other forms.
 7. A pharmaceutical compositioncomprising the polymorph of claim 1 and further comprising one or morepharmaceutically acceptable ingredients selected from the groupconsisting of carriers, excipients, diluents, additives, fillers,lubricants and binders.
 8. The pharmaceutical composition of claim 7,wherein the composition is formulated for oral administration.
 9. Amethod of producing crystal polymorphic Form VI of ST-246, comprisingthe steps of: a) dissolving ST-246 in at least one solvent to make asolution; b) cooling said solution to a temperature that causes thepreferential crystallization of said ST-246 polymorphic Form VI; and c)optionally drying the formed crystals of ST-246, wherein said solvent isselected from the group consisting of nitromethane, methanol andchloroform.
 10. The method of claim 9 further comprising adding seedcrystals of polymorphic Form VI ST-246 during step (b).
 11. The methodof claim 9, wherein said solvent does not contain water.
 12. The methodof claim 9, wherein said solvent is nitromethane.
 13. A dosage unit formfor oral administration comprising the pharmaceutical composition ofclaim 8, wherein ST-246 has a D90% particle size diameter of up to about300 microns.
 14. A dosage unit form according to claim 13, wherein saidST-246 has a D90% particle size diameter of about 5 microns.
 15. Adosage unit form according to claim 13, wherein said ST-246 has a D90%particle size diameter of about 16.6 microns.
 16. A dosage unit formaccording to claim 13, wherein said ST-246 has a D90% particle sizediameter of about 26.6 microns.
 17. A dosage unit form according toclaim 13, wherein said ST-246 has a D90% particle size diameter of about75 microns.
 18. A unit dosage form for oral administration comprising:(a) about 200 mg of ST-246, wherein ST-246 is ST-246 polymorph Form VI;(b) about 33.15 mg of lactose monohydrate; (c) about 42.90 mg ofcroscarmellose sodium; (d) about 1.95 mg of colloidal silicon dioxide;(e) about 13.65 mg of hydroxypropoyl methylcellulose; (f) about 7.8 mgof sodium lauryl sulfate; (g) about 1.95 mg of magnesium stearate; and(h) a quantity of microcrystalline cellulose up to about 88.60 mg suchthat the total weight of the dosage form is about 390 mg.